the divergence, actions, roles, and relatives of [PDF]

Physiol Rev 93: 803–959, 2013 doi:10.1152/physrev.00023.2012

THE DIVERGENCE, ACTIONS, ROLES, AND RELATIVES OF SODIUM-COUPLED BICARBONATE TRANSPORTERS Mark D. Parker and Walter F. Boron Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio

L

I. INTRODUCTION

Roos and Boron (811). In 2003, Chesler (186) focused on pH regulation in the brain. This journal reviewed vacuolar H⫹ pumps in the contributions by Forgac in 1989 (292), by Nelson and Harvey in 1999 (678), and by Wagner et al. in 2004 (1015). The journal considered H-K pumps in the effort of Hersey and Sachs in 1995 (380). Na-H exchange was the subject of the 1997 review by Wakabayashi and co-workers (1017). Recently, Lee et al. (555) have examined HCO3⫺ secretion by the pancreas and salivary glands (555). However, Physiological Reviews has not examined HCO3⫺ transporters per se.

A. Regulation of pH

B. Scope of This Review

pH is one of the most important parameters for life. Virtually every biological process is sensitive to changes in pH, and some are exquisitely sensitive. Thus transporters have evolved to regulate pH in organelles, the cytosol, and the extracellular fluid. Not surprisingly, dysregulation of pH is associated with a wide array of pathologies (TABLE 1), including cancer, hypertension, reperfusion injury, amyloid deposition (e.g., in Alzheimer’s disease), and aging.

The movement of bicarbonate equivalents, HCO3⫺ itself, CO32⫺, or the NaCO3⫺ ion pair, across the plasma membrane is an integral part of the regulation of pHi and the transepithelial transport of solutes and fluid. Disturbances in HCO3⫺ transporter genes are associated with a variety of pathologies and can potentially impact any of the vast array of pH-sensitive proteins and processes summarized in TABLE 1.

The transporters responsible for pH regulation in various compartments include vacuolar-type ATPases or H⫹ pumps, gastric-type H⫹-K⫹-ATPases or pumps, Na-H exchangers, and bicarbonate (HCO3⫺) transporters. Physiological Reviews last appraised the general subject of intracellular pH (pHi) regulation in 1981, with the review by

Bicarbonate transport in animals is effected by the eight physiologically distinct mechanisms numbered 1– 8 in the generic epithelial cell in FIGURE 1.

I. II. III. IV. V. VI. VII. VIII.

INTRODUCTION NCBT EMERGENCE AND DIVERGENCE NCBTs AND RELATIVES IN NONMAMMALS GENERAL FEATURES OF NCBTs NCBTs IN MAMMALS RELATIVES OF NCBTs IN MAMMALS CONCLUDING REMARKS APPENDICES

803 810 819 836 845 912 925 931

1) Conductive HCO3⫺ transport mediated by anion permeable channels such as GABA- and glycine-gated anion chan-

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Parker MD, Boron WF. The Divergence, Actions, Roles, and Relatives of SodiumCoupled Bicarbonate Transporters. Physiol Rev 93: 803–959, 2013; doi:10.1152/physrev.00023.2012.—The mammalian Slc4 (Solute carrier 4) family of transporters is a functionally diverse group of 10 multi-spanning membrane proteins ⫺ that includes three Cl-HCO3 exchangers (AE1–3), five Na⫹-coupled HCO3 transporters (NCBTs), and two other unusual members (AE4, BTR1). In this review, we mainly focus on the five mammalian NCBTs-NBCe1, NBCe2, NBCn1, NDCBE, and NBCn2. Each plays a specialized role in ⫺ maintaining intracellular pH and, by contributing to the movement of HCO3 across epithelia, in maintaining whole-body pH and otherwise contributing to epithelial transport. Disruptions involving NCBT genes are linked to blindness, deafness, proximal renal tubular acidosis, mental retardation, and epilepsy. We also review AE1–3, AE4, and BTR1, addressing their relevance to the study of NCBTs. This review draws together recent advances in our understanding of the phylogenetic origins and physiological relevance of NCBTs and their progenitors. Underlying these advances is progress in such diverse disciplines as physiology, molecular biology, genetics, immunocytochemistry, proteomics, and structural biology. This review highlights the key similarities and differences between individual NCBTs and the genes that encode them and also clarifies the sometimes confusing NCBT nomenclature.

MARK D. PARKER AND WALTER F. BORON

Table 1. The importance of pH regulation Process

Pathological Associations

Cell survival

Acid-extruding mechanisms defend intracellular pH from catastrophic, pro-apoptotic acidosis (e.g., Ref. 112). However, acidosis is anti-apoptotic for some cells (e.g., Refs. 978, 1057). Telomere structure is pH sensitive (417).

Tumor proliferation: In cancer cells, enhanced acid-extrusion ability and a lowering of local extracellular pH, contributing to an acidic, tumorpermissive environment while defending tumor pHi (546, 935). Autophagy is reduced at acidic extracellular pH (1058). Heart failure: Hypoxia in combination with acidosis is pro-apoptotic in cardiac myocytes (519).

Na⫹ homeostasis

NCBTs and NHEs are secondary active transporters that couple acid extrusion with Na⫹ influx, thereby contributing to regulation of [Na⫹]i and plasma [Na⫹]. ENaC activity is modulated by ⫺ pH and [HCO3 ] (163, 196, 730).

Cell migration and Wound healing

Acid-extruders act as plasma membrane anchors for cytoskeletal components (e.g., Ref. 243) and can promote an isosmotic volume increase at the leading edge of migrating cells (910). Acid extrusion promotes wound healing (1062) as well as dendritic spine growth (249).

Solute transport

Many solute carriers such as H⫹-coupled amino acid transporters (95) influence or are influenced by pH. Furthermore, acid-base status influences the expression of other, nominally pH-independent carriers (660, 688).

Protein folding/assembly

The stability and conformation of almost all proteins is pH dependent, due to electrostatic effects (946). Consequently, the oligomeric state of diverse proteins (e.g., Refs. 145, 838, 1084) as well as interactions between protein binding partners (e.g., Refs. 661, 687) can be pH dependent.

Reperfusion injury: The influx of Na⫹ that accompanies enhanced acid extrusion following ischemia can tend to reverse Na⫹-Ca2⫹ exchangers, causing a pathological increase in [Ca2⫹]i (956, 993, 997). Hypertension: ⫺ Dysregulation of H⫹ and HCO3 transporters is associated with hypertension (89, 92, 1020). Tumor metastasis: Acidosis, by stimulating the acid-extruding activity of NHE1, can promote metastasis of tumor cells (151, ⫺ 547). HCO3 , in its capacity as a buffer, is inhibitory to metastasis (410, 801). Drug sensitivity: Acid-base status can influence the efficacy and toxicity of drugs (647, 705) and acidosis induces drug resistance in tumors via activation of Pglycoprotein (963). Amyloidosis: Acidosis promotes amyloid formation (294, 395, 784, 815, 936), potentially impacting the severity of Alzheimer’s Disease and scrapie. Carcinogenesis: The stability of the tumor-suppressing tetrameric form of a mutant p53 is readily destabilized by mild alkalosis, a mechanism suggested to underlie the increased incidence of carcinomas in individuals who carry this mutation (250).

Protein glycosylation

An acidic environment in the Golgi is crucial for appropriate localization of glycosyltransferases and therefore for N-glycosylation of proteins (799). Some interactions between proteins and the plasma membrane or between proteins and cell-surface receptors are pH dependent (e.g., Refs. 255, 370).

Interactions at the cell surface

Amyloid deposition: Deposition of amyloids is enhanced at acidic pH (131, 171, 513, 785). Viral infection: The fusion of viral particles with the host plasma membrane is pH dependent, although the direction of the dependence may vary between viruses (e.g., Refs. 363, 548, 751, 803, 1046). Bacterial colonization: The colonization of H. pylori on the surface of gastric mucosa is enhanced at acidic pH (787). Moreover, in a porcine model of cystic fibrosis, the acidity of airway surface liquid diminishes its antimicrobial properties (745).

Continued

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pH-Dependent Physiology

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

Table 1.—Continued Process

pH-Dependent Physiology ⫺ Sensors for acid, alkali, and CO2/HCO3 (129, 181, 1105, 1107) are expressed in multiple cell types, mediating the cellular effects of acid-base status. Furthermore, numerous receptor/ligand interactions are influenced by pH (e.g., Refs. 227, 295, and 691).

DNA and protein synthesis and stability

Incorporation of amino acids into polypeptides is reduced under acidic conditions (451, 736). pH-responsive elements in certain RNAs confer increased lifetime to those transcripts in acidosis (409). Excessive neuronal firing can reduce neuronal pH and in turn, neuronal excitability is reduced in response to lowering extracellular and intracellular pH (186, 187, 783). Most K⫹ channels are pH dependent (e.g., Refs. 67, 424, 1053). NCBTs play critical roles in defending neuronal pHi and regulating the pH of the neuronal microenvironment (via their action in astrocytes and choroid plexus epithelia). ⫺ The fluid movement that follows HCO3 transport maintains the clarity of the cornea (96) and lens (65) and also maintains retinal attachment (400, 534). In the inner ear, low endolymph pH can reduce response of hair cells to auditory stimuli (150).

Neuronal excitability

Special senses

Muscle contraction

Bone remodeling

Digestion

Multiple elements of excitation-contraction coupling in cardiac, smooth, and skeletal muscle are inhibited at low pH including neurotransmitter release (586), gap junction conductivity (379, 707), as well as the action of the contractile apparatus (e.g., Refs. 286, 497, 892, 1045). Bone remodeling requires H⫹ secretion (62) and ⫺ HCO3 resorption (797), thus bone maintenance is exquisitely pH sensitive. Furthermore, osteoclast survival is reduced by acidosis (e.g., Ref. 112).

Enamel formation (456), saliva secretion (555), enzymatic digestion, and mucosal protection ⫺ (17) are all pH/HCO3 -dependent processes.

Type 2 diabetes mellitus: Elevated ⫺ serum HCO3 was associated with a reduced risk of developing type 2 diabetes in a study of 650 women (625). Tumor proliferation: Expression of the acid sensor TDAG8 in tumor cells enables the cells to adapt to the extracellular acidic environment (415). Anxiety disorders: Acidosis and detection of H⫹ by the acid sensor ASIC-1a elicits acquired fear behavior. Overexpression of ASIC-1a in mice is a model of anxiety (204, 205, 1032, 1117).

Altered neuronal excitability: Disruption of NCBT genes is associated with autism, epilepsy, mental retardation, and migraine (360, 411, 516, 830, 930).

Loss of vision: Mutations in acidbase transporters are associated with cataracts, glaucoma, and retinopathy (e.g., Refs. 30, 93, 411). Acidosis induces retinopathy in neonatal rats (391, 392). Loss of hearing: Mutations in acid-base transporters are associated with hearing loss (e.g., Refs. 93, 473). Paralysis: Lactic acidosis (e.g., Ref. 85) and renal tubular acidosis (e.g., Ref. 119) result in muscle weakness.

Bone remodeling defects: H⫹ secretion defects in osteoclasts are associated with osteopetrosis (e.g., Refs. 455, 866), whereas whole-body acidosis can be associated with bone dysplasia (e.g., Refs. 313, 602). Poor dentition: Defects in acid-base transporters result in defective enamel deposition (540, 617). Ulceration: Metabolic and respiratory acidoses increase the incidence of gastric lesions (142, 507). Gut lumen pH is unusually acidic in some individuals with ulcerative colitis (690). Diarrhea: Dysregulation of acid-base transport can result in decreased nutrient absorption, increased fluid secretion, and diarrhea (388, 938, 1092).

Continued

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Cell signaling

Pathological Associations

MARK D. PARKER AND WALTER F. BORON

Table 1.—Continued Process

pH-Dependent Physiology

Immune response (544)

Extracellular acidosis activates neutrophils (978) but reduces TNF-␣ secretion by alveolar macrophages (82). Superoxide production by NADPH oxidase during the respiratory burst is accompanied by a decrease in pHi that is countered by the action of H⫹ channels (230).

Fertility

Multiple aspects of male and female fertility, including sperm maturation and cervical mucus ⫺ release are influenced by pH and HCO3 (597, 665).

Pathological Associations

Tumor proliferation: The reduction of macrophage cytotoxicity in the acidic tumor microenvironment would promote tumor survival (82). Immunodeficiency: Inability to defend macrophage pHi during respiratory burst might reduce the ability of macrophages to counter bacterial infection (discussed in Ref. 230). Reduced fertility: Mice with ⫺ disrupted HCO3 transporters are sub- or infertile (e.g., Refs. 165, 389, 638).

nels (98, 460), the cystic fibrosis transmembrane conductance regulator CFTR (752), ClC channels (827), and Ca2⫹-activated chloride channels (776, 777).1 2) Apical Na⫹-independent Cl-HCO3 exchange, effected by anion exchangers encoded by members of the solute carrier 26 (Slc26) gene family (Slc26a3, Slc26a4, Slc26a6, and Slc26a9), reviewed in References 153, 259, and 888.2 3) A basolateral Na ⫹ -independent SO 4 -2HCO 3 exchanger, or oxalate-2HCO 3 exchange encoded by Slc26a1 (474, 517, 525). 4) Electroneutral K/HCO3 cotransport. The molecular identity of the responsible protein(s) has yet to be established (386, 387, 570, 1097). 5) Basolateral Na⫹-independent Cl-HCO3 exchange, mediated mainly by the electroneutral anion exchangers AE1 1 In most cases, these channels are at best poorly permeable to ⫺ HCO3 and, in most cases, the physiological significance of this permeability is not demonstrated. 2 For the purposes of this review, we depict Slc26a3, -4, -6, -7, and -9 as electroneutral Cl-HCO3 exchangers; in fact, the molecular action of these Slc26 transporters is controversial. Slc26a3 has been described as being capable of electroneutral Cl-HCO3 exchange by some (26, 908) but electrogenic 2Cl-HCO3 exchange by others (499, 871). Slc26a4 is capable of electroneutral Cl-HCO3 exchange (872), a description that is uncontested. Slc26a6 has been described as being capable of electroneutral Cl-HCO3 exchange by some (185) but electrogenic Cl-2HCO3 exchange by others (499, 871, 1052). Slc26a7 has been described as an electroneutral Cl-HCO3 exchanger (743), and also as a pH-sensitive Cl⫺ channel with no anion exchange activity (488). Slc26a9 has been decribed as a Cl-HCO3 exchanger of undetermined electrogenicity/electro⫺ neutrality (1055), an electrogenic nCl-HCO3 exchanger with a HCO3 ⫺ independent Cl⫺ conductance (168), a HCO3 -independent Cl⫺ chan⫺ nel with no anion exchange activity (258), and also as a HCO3 stimulated Cl⫺ channel with no anion exchange activity (609). The reasons underlying the apparent disparities among studies have not been determined.

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(Slc4a1),3 AE2 (Slc4a2), and AE3 (Slc4a3) and perhaps some members of the Slc26 family (e.g., Slc26a7). 6) Electrogenic Na/HCO3 cotransport, mediated by NBCe1 (Slc4a4) and NBCe2 (Slc4a5), which are predicted to operate with varying stoichiometry in different cell-types (6a versus 6b in FIGURE 1). 7) Electroneutral Na/HCO3 cotransport, mediated by NBCn1 (Slc4a7) and NBCn2 (Slc4a10). 8) Na⫹-driven Cl-HCO3 exchange, mediated by NDCBE (Slc4a8). Groups 5– 8 include members of the Slc4 family that, in vertebrates, are normally located in the basolateral (or equivalent) membranes of polarized cells, in some instances complementing the usually apical (or equivalent) distribution of certain HCO3⫺-transporting Slc26 family members. Groups 6 – 8 are collectively referred to as Na⫹-coupled bicarbonate transporters (NCBTs) and are the major focus of the present review. The general predicted topology of mammalian, and likely all vertebrate, Slc4s is exemplified by the depiction of human NBCe1 in FIGURE 2A. Typically, each Slc4 protein has a large NH2-terminal (Nt) cytoplasmic domain, followed by a large multi-spanning transmembrane domain (TMD)

3 Following the recommendations of The HUGO (Human Genome Organization) Gene Nomenclature Committee (HGNC), we use the terms “SLC4” (gene) and “SLC4” (gene-product) only in instances when we are specifically and exclusively referring to human genes and products. The terms “Slc4” and “Slc4” are used in reference to vertebrate genes/products in general (even if that grouping includes humans), whereas “Slc4-like” and “Slc4-like” refer to related nonvertebrate genes. The common names AE, NBC, and NDCBE are capitalized throughout, independently of parent organism.

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Proteins, processes, and pathologies in mammals that are influenced by or that influence pH. Processes and diseases that are specifically related to NCBT function and dysfunction are discussed in detail in later sections of the review.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Apical

Tight junction

Basolateral

–

HCO3– channel

HCO3–

2 HCO3

1

3

4

Cl–

5

K+ HCO3– HCO3–

Basolateral SO42––2 HCO3– exchanger

Electroneutral K+/HCO3– cotransporter

Basolateral Cl––HCO3– exchanger

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6a

SO42–

Na+ 3 HCO3– Electrogenic Na+/HCO3– cotransporters

Na+ Apical Cl–-HCO3– exchanger

HCO3–

2

Cl–

2 HCO3– Na+ HCO3–

6b

Electroneutral Na+/HCO3– cotransporter

7

Na+ 8 2 HCO3–

Cl–

Electroneutral Na+–driven Cl–-HCO3– exchanger

FIGURE 1. Functional classifications of bicarbonate transporters. Diagram of a generic epithelial cell show⫺ ing the typical subcellular distribution of the 8 classes of HCO3 transporters. Anion channels (1) and anion ⫺ exchangers of the Slc26 family (2) perform HCO3 secretion across the apical membrane. Basolateral Slc26a1 ⫺ functions as SO42--2HCO3 exchanger (3). An as yet unknown transporter (4) is presumed to be responsible for a basolateral K/HCO3 cotransport activity in the inner medulla. Members of the Slc4 family (5–8) are usually located in the basolateral membranes of polarized epithelia. Cl-HCO3 exchangers (5) and electrogenic Na/ ⫺ HCO3 cotransporters with a calculated 1:3 stoichiometry (6a) act as acid-loaders, supporting HCO3 absorption into the blood. Electrogenic Na/HCO3 cotransporters with a 1:2 stoichiometry (6b), electroneutral Na/HCO3 ⫺ cotransporters (7), and Na⫹-driven Cl-HCO3 exchangers (8) act as acid-extruders, supporting HCO3 secretion across the apical membrane by transporter classes 1–2.

that includes one glycosylated extracellular loop, and concludes with a shorter COOH-terminal cytoplasmic domain (Ct). As depicted in FIGURE 2B, nonvertebrate Slc4-like (see footnote 3) products, such as those from bacteria, fungi, amoebas, and plants, are predicted to retain the same general topology but to have shorter Nts and to have extracellular loops of varying lengths. The molecular identity of the Na⫹-independent Cl-HCO3 exchangers AE1-AE3 (included in group 5, above) has been known for some time. It is more than 30 years since AE1

was first demonstrated to be the erythrocyte anion transporter (1040). The cloning of the murine Slc4a1 cDNA that encodes AE1 was reported in 1985 (510) and was soon followed by the discovery and cloning of Slc4a2 (23, 242) and Slc4a3 (509) products. These three genes appeared to be the extent of the gene family until 1997, when Romero et al. (809) published the cDNA and the elucidated protein sequence of an electrogenic Na/HCO3 cotransporter from the tiger salamander, Ambystoma tigrinum. Electrogenic Na/HCO3 cotransport had first been described in the salamander proximal tubule (PT) by Boron and Boulpaep 14

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A EL3 C C

TMD

C C

1

2

3

4

5

Lumen

6

7

8

9

10

11

E 13

14

Cytosol Ct

Nt

H2N

B

TMD

Nt 1 23 4 5

EL3

6 7

9

1011 1314

E (human NBCe1-A; 1,035 aa)

Mammalian

(bacterial ‘Nitro’; 513 aa)

Bacterial

Fungal

Plantal

8

Ct

(yeast Bor1p; 576 aa)

100 aa

(thale cress AtBOR1; 729 aa)

(slime mold Slc4-like product; 768 aa)

Amoebal

FIGURE 2. Presumed topology of NCBTs and Slc4-like transporters. Presumed topology of the electrogenic Na/HCO3 cotransporter NBCe1 (A), representing a probable common structure for all five mammalian NCBTs and most nonmammalian NCBTs. The model shows the extended cytosolic amino- and carboxy-terminal domains (Nt and Ct) linked via a transmembrane domain (TMD) that includes 14 transmembrane spans (TMs), one of which is thought to be an extended region (E) rather than an ␣-helix. In mammalian NCBTs, the third extracellular loop (EL3) between TMs 5 and 6 usually includes multiple cysteine residues (C) and multiple putative glycosylation sites (Y). A sequence alignment displaying these features for human NCBTs is provided in Appendix I. Pictorial depiction of NBCe1 domain sequences aligned against homologous regions of nonvertebrate Slc4-like transporters (B). Horizontal purple bars represent protein sequence laid out from Nt to Ct. Gaps in sequence alignment are depicted as horizontal lines. Vertical bars represent position of ␣-helical TMs. Note the shorter Nt and EL3 in nonvertebrates. The amoebal Slc4-like transporter includes an extended Nt (yellow region), but it shares no significant sequence identity with the extended Nt of vertebrate Slc4s. The sequence of nonmammalian Slc4-like protein is provided in Appendix II.

years earlier (103), and the cloning of the responsible gene product allowed sequence comparisons that importantly demonstrated that NCBTs were members of the same Slc4 family as AE1–3. The salamander cDNA reported by Romero et al. is now recognized as the archetypal Slc4a4 gene product. Work from several groups then revealed the existence of six further members of the vertebrate Slc4 gene family (337, 720, 765, 767, 982, 1021), bringing the total

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number to 10. These novel genes were designated Slc4a5 and 7–11 (Slc4a6 was rescinded, see below). The products of 5 of these 10 Slc4 genes (NBCe1, NBCe2, NBCn1, NBCn2, and NDCBE) have demonstrated NCBT activity. The function of AE4, the product of Slc4a9, is controversial, but it is reported to mediate Cl-HCO3 exchange in some heterologous systems. Bicarbonate transporter related protein 1 (BTR1), the product of Slc4a11, likely mediates

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HOOC

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

borate transport, a function common to the Slc4-like transporters (the BORs) of some fungi and higher plants. The values in TABLE 2 and the unrooted phylogenetic tree in FIGURE 3 summarize the relatedness, at the level of protein sequence, of the TMDs of human SLC4s. Note that SLC4 function follows sequence relatedness. The first major sequence classification corresponds to AEs (red group) versus NCBTs/AE4 (gray group) versus BTR1. The second major sequence classification corresponds to electrogenic NCBTs (blue group) versus electroneutral NCBTs (red group).

BTR1

Putative Boron Transporter

(SLC4A11)

AE3 (SLC4A3)

Cl-HCO3 Exchangers

AE2 (SLC4A2)

AE1 (SLC4A1)

Electroneutral Na/HCO3 Cotransporters AE4 (SLC4A9)

NBCn2 (SLC4A10) 0.1

NDCBE

NBCe1 (SLC4A4)

(SLC4A8)

NBCn1 (SLC4A7)

NBCe2

(SLC4A5)

C. Review Outline

Electrogenic Na/HCO3 Cotransporters

In section III we review the actions and roles of NCBTs and Slc4-like transporters in nonmammalian species. In addition to being of interest to comparative physiologists, this discussion brings together, for the first time, data that provide insight into how the actions and roles of Slc4-like proteins have evolved to their present status in mammals. In section IV, we look at the structural features/domains of a typical mammalian NCBT. Here we present a second way to consider the structural relation between NCBTs: an analysis of conserved and variable protein regions. We also present a summary of maneuvers known to inhibit or stimulate mammalian NCBTs.

Table 2. Relatedness among human SLC4 protein sequences

FIGURE 3. Relatedness among human SLC4s. The unrooted phylogram displays the relatedness at the level of protein sequence among the transmembrane domains of the 10 human SLC4 proteins. Note how transporter function correlates with protein sequence similarity. The phylogram was generated using ClustalW (183) and TreeView (704). A sequence alignment of the 10 human SLC4s is provided in Appendix I, and the protein sequence identity among the transmembrane domains of human SLC4s is provided in TABLE 2.

In section V we then consider, in turn, each of the 5 mammalian NCBTs and, for each, 10 categories of key characteristics. The italicized terms below correspond to the titles of the headings in section V. A) Summary. A précis of the key characteristics, actions, and roles for each NCBT, serving as a quick reference guide for the casual reader. B) Nomenclature. A definitive guide to the naming of each NCBT, necessary because nonstandard and redundant nomenclatures have made collation and interpretation of the literature confusing. In each case we link the nomenclature used in this review with a GenBank sequence accession number. C) Molecular action. A detailed account of the substrates and transport modes of each NCBT.

NBCe1 NBCe2 NBCn1 NBCn2 NDCBE AE4 BTR1

AE1 AE2 AE3 NBCe1 NBCe2 NBCn1 NBCn2 NDCBE AE4 BTR1

39 42 42 100

38 39 39 71 100

38 41 42 57 50 100

38 40 42 58 55 81 100

39 41 41 58 54 81 84 100

38 43 43 62 58 52 52 52 100

28 30 30 28 28 28 29 30 29 100

Percentage identities among the protein sequences of human SLC4s transmembrane domains. Identities were computed by pairwise BLAST (951). AE1–AE3 share 50 – 60% identity within their transmembrane domains. Alignments of human NCBT protein sequences are provided in Appendix I, and GenBank protein accession numbers are provided in Appendix IV.

D) Genome. A summary of the key features of the genes encoding each NCBT. E) Structural features and variants. A definitive guide to the known products created from each NCBT gene. F) Distribution. A comprehensive detailing of the localization of NCBT transcripts and proteins from the intracellular to the whole organ level. G) Physiological roles. A review of the known and speculated physiological roles of each NCBT in specific tissues. H) Causes of upregulation. A consideration of the perturbations that result in upregulation of NCBT at the level of transcript/protein abundance or activity.

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In the first major part of our review, section II, we examine the NCBTs and Slc4-like genes from bacteria, fungi, plants, and animals and consider how the Slc4 gene family has diverged from a single common ancestor into the 10 members that we recognize today, including the 5 mammalian NCBTs. In addition, we examine the genealogy of extant vertebrate NCBT genes based on an analysis of conserved exon boundaries. Section II should be valuable to those interested in any Slc4 protein.

MARK D. PARKER AND WALTER F. BORON I) Causes of downregulation. A consideration of the perturbations that result in downregulation of NCBT at the level of transcript/protein abundance or activity. J) Consequences of dysfunction. A review of the diverse pathological states associated with defects and variations in NCBT genes and products. Characteristics G–J, taken together, provide an integrated picture of the importance of each NCBT.

In section VII, our final section entitled “Concluding Remarks,” we draw together from Section V several recurring themes, unresolved issues, and emerging topics in the NCBT field. Throughout the review we summarize critical information, for quick reference, in the form of Tables. Here the reader will find guides to the importance of pH regulation, the relatedness among Slc4 and Slc4-like gene products, NCBT inhibitors, NCBT distribution, and pathological mutations in the SLC4A4 gene. In our Appendices, we complement the content of the review with detailed information, such as complete sequence alignments of the NCBT proteins and their splice variants. We also present tables of GenBank protein accession numbers of all of the Slc4 and Slc4-like gene products and variants discussed in this review to allay confusion about nomenclature. The accession numbers are hyperlinked to the National Center for Biotechnology Information (NCBI) database for ease of reference. We also present some additional data about NCBT distribution, namely: 1) an NCBT expression pattern in humans and mice inferred from a tabulation of the origins of NCBT expressed-sequence tags deposited on a public database; 2) a discussion of “antiNBC3” immunoreactivity, which discloses a distribution pattern for NBCn1 (Slc4a7) that is different from that suggested by other probes; and 3) a discussion of several apparently conflicting reports of AE4 (Slc4a9) localization within the mammalian kidney. These last two appendices will be useful for those who seek to make sense of the often conflicting data concerning the distribution of these proteins. This review is not intended to focus on the regulation of pHi per se, although the NCBTs play key roles in this task.

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II. NCBT EMERGENCE AND DIVERGENCE A. Summary In this section we consider how the five mammalian NCBT genes emerged from a single primordial Slc4-like gene. As we shall see, gene and genome duplications as well as gene losses have resulted in the inclusion of a diverse number of Slc4-like genes in the genomes of diverse organisms. Fungal and plantal Slc4-like genes predominantly encode boron transporters. In the animal lineage, distinct NCBT-like genes appeared no later than the emergence of Eumetazoa such as sea anemones. The most primordial Slc4-like geneproduct with NCBT activity is the Na⫹-driven anion/bicarbonate exchanger ABTS-1 from the nematode worm, Caenorhabditis elegans. The genome of the chordate sea squirt Ciona intestinalis includes three Slc4-like genes, one of which shares a single common ancestor with the five mammalian NCBTs and “AE4.” The emergence of individual NCBTs was initiated by the divergence of an NBCe1/ NBCe2/“AE4” ancestor from an NBCn1/NDCBE/NBCn2 ancestor. The five mammalian NCBTs were probably distinct entities no later than the emergence of primordial vertebrates such as lampreys.

B. Emergence, From an Ancestral Prokaryote to Early Chordates, of AE-like, NCBT-like, and BOR-like Genes The recent proliferation of genome sequence data, backed up by the physiological characterization of certain products, allows us to begin to appreciate the diversity of Slc4 and Slc4-like genes and products. In FIGURE 4, which gives examples of the major taxonomic divisions, we represent the taxonomic relationship of diverse organisms along with their known complement of Slc4-like genes. Some of these products have demonstrated Cl-HCO3 exchanger (AE), Na-coupled HCO3 transporter (NCBT), or borate trans-

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In section VI we provide a similar, though abbreviated, consideration for the three AEs (AE1–3) and the related products AE4 and BTR1. Section VI, A and B, with their organization of the NCBT literature in light of the wealth of AE data, will be of particular value to those new to the larger Slc4 field. Our consideration of AE4 and BTR1, which are of interest to the NCBT community, are the first detailed reviews of these unusual family members.

Rather, the reader is referred to the review by Roos and Boron (811), the more recent chapter by Bevensee et al. (77), or the analysis of Boron (101). Likewise, the present review does not focus on the kinetics or thermodynamics of HCO3⫺ transport, for which we would recommend References 339 and 529. For a more concise overview of NCBTs, we direct the reader to recent reviews in 2004 by Romero et al. (807), in 2006 by Pushkin and Kurtz (772), in 2007 by Parker and Boron (714), and in 2009 by Casey and Cordat (153), Romero et al. (805), and Boron et al. (104). We intend this document to provide a clear review of NCBT genes and proteins for those new to the field, as well as an up-to-date and comprehensive reference resource for Slc4 researchers. Note that meta-analyses and reinterpretations of published data that do not include a link to a published article are the opinions of the authors.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Prokaryota Nitrifying bacterium Nitrococcus mobilis

Eukaryota

ZP_01128815

Primitive

Haptophyceae

Amoebozoa

Marine Phytoplankter* Emiliania huxleyi

Plants

Multiple fragments** fra ag

Primitive 7

Basidiomycota* Inky-cap mushroom Coprinopsis cineria EAU85942

BOR-like 15

Porifera Sponge* Suberites domuncula CAF32326 (NBCSA)

AE-like

9

BOR-like 3G

Placozoa Tablet animal* Trichoplax adhaerens XP_002110840

AE-like

9

XP_002110841

AE-like

16

XP_002115332/3

XP_001765760

Primitive 13

o na otin na Pezizomycotina

Bilateria

Moss* Physcomitrella patens

Tracheophyta

Bryophyta

Saccharomycotina

Black mold n i err Aspergillus nig niger

Brewer’s Yeast Saccharomyces cerevisiae

BOR-like 17

Cnidaria Starlet sea anemone* Nematostella vectensis

CAK43550

BOR-like 6G

XP_001623893

AE-like

8

XP_001766018 (PpBOR1) BOR-like 29

CAK39400

O 14 BOR-like

XP_001637907

AE-like

11

XP_001759676 (PpBOR2) BOR-like 28

CAL00656

BOR-like 23

XP_001619189**

NCBT-like 17

XP_001632673** taken with XP_001632674**

BOR-like 24

Monocotyledons

Thale cress Arabidopsis thaliana

Rice Oryza sativa

75

Pseudocoelomata Nematode worm Caenorhabditis elegans

NP_001078071 (AtBOR1)

BOR

69

NP_001067049 (OsBOR1)

BOR

NP_191786 (AtBOR2)

BOR

59

ABD78950 (OsBOR2)

BOR-like 30

NP_001024827 (ABTS-4)

AE-like

6G

NP_187296 (AtBOR3)

BOR

45

BAG92966 (OsBOR3)

BOR

27

NP_492258 (ABTS-1)

NCBT

21

NP_172999 (AtBOR4)

BOR

27

ABD78951 (OsBOR4)

BOR-like 30

NP_509936 (ABTS-2)

BOR-like 14

NP_177619 (AtBOR5)

BOR

24

NP_001033333 (ABTS-3)

BOR-like 18

NP_197925 (AtBOR6)

BOR-like 25

NP_194977 (AtBOR7)

BOR-like 24

Mollusca

49

BOR

Coelomata

Eudicotyledons

NP_014124 (Bor1p)

Protostomia

Deuterostomia

Panarthropoda

Longfin Inshore Squid*

Echinodermata

Doryteuthis (Loligo) pealei

Chordata

Sea urchin* Strongylocentrotus purpuratus

Fruit fly Drosophila melanogaster

Sea squirt* Ciona intestinalis

ABF06445

AE-like

16

NP_648357 (CG8177)

‘AE-like’ 15

XP_793649**

‘AE-like’

10

XP_002124608

ABF06444 (sqNBCe)

NCBT

18

NP_523501 (NDAE1)

NCBT

XP_791964

‘AE-like’

16

XP_002124093

NCBT-like 66

AAN75454 (sqNDCBE)

NCBT

22

NP_001073019 (Sp-NBC)

‘NCBT-like’ 21

XP_002128564

BOR-like 73

XP_786033

‘NCBT-like’ 22

XP_784629

‘BOR-like’

18

AE-like

66

15

FIGURE 4. Diversity of nonvertebrate Slc4-like transporters. The cladogram represents the evolutionary relationships of a variety of organisms, based on the taxonomy defined by The Tree of Life Web Project (http:// tolweb.org) and the taxonomy database at NCBI (839). Each organism is represented by a boxed table that includes, in the top row, the common and scientific name of the organism. If the full genome sequence of the organism has not been published, the organism’s name is noted with an asterisk. The remaining rows of each table provide information about the Slc4-like proteins that are encoded by the genome of each organism. These rows are divided into three columns: Column 1 lists the GenBank protein accession number and, where appropriate, common name for each Slc4-like protein. Partial sequences are marked with a double asterisk. Column 2 lists the function of each transporter (AE, NCBT, or BOR) or, if unknown, the assignment of each protein to one of the four groups (AE-like, NCBT-like BOR-like, or Primitive) defined in text that indicates their relatedness to their reference sequences for each kingdom (red text). Column 3 shows a numerical “divergence score” (DS, see text) denoting the extent of similarity to the reference protein. Proteins that do not bear a strong similarity to any one of the reference proteins are marked with a “G” for “generic.” A hyperlink to the protein sequence of each transporter is provided in Appendix II.

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Primitive 2G

XP_646790

Eumetazoa

XP_001417272 XP_001417844

3-5G

Ascidiomycota

Green alga cim mari m rinus’ Ostreococcus ‘luc ‘lucimarinus’

Embryophyta a

a Chlorophyta

Primitive

Social amoeba Dictyostelium discoideum

Animals

Fungi

MARK D. PARKER AND WALTER F. BORON

1) “Primitive” (present only in bacteria and plants): most resembling the bacterial Slc4-like transporter that we have provisionally termed “Nitro” (see “Primitive” in FIGURE 4/Plants and FIGURE 5). 2) AE-like (present only in animals): most resembling the sea-squirt protein that shares a common ancestor with all vertebrate Na⫹-independent Cl-HCO3 exchangers (see Ciona “AE-like” in FIGURE 4/Sea Squirt). AE-like transporters cluster with the Ciona AE-like reference protein on

“BOR” Group II

AtBOR4* OsBOR2 OsBOR3*

an unrooted phylogenetic tree (FIGURE 6) and exhibit a characteristic “fingerprint” of sequence identity inasmuch as they are more similar to NCBTs than BORs. 3) NCBT-like (present only in animals): most resembling the sea-squirt protein that shares a common ancestor with all vertebrate Na⫹-coupled HCO3⫺ transporters (see “NCBT-like” in FIGURE 4/Sea Squirt). NCBT-like transporters cluster with the Ciona NCBT-like reference protein on an unrooted phylogenetic tree (FIGURE 6) and exhibit a characteristic “fingerprint” of sequence identity inasmuch as they are more similar to AEs than BORs. Of course, all invertebrate Slc4-like transporters with demonstrated Na⫹coupled HCO3⫺ transport function fall into this category. 4) BOR-like: because borate transporter proteins share little identity across kingdoms (22–27%; see TABLE 3), we define “BOR-like” as follows. For plants, most resembling the established borate transporter of thale cress (see “AtBOR1” in FIGURE 4/Thale Cress) than our bacterial Slc4-like reference protein “Nitro.” Of course, all plantal Slc4-like transporters with demonstrated borate transport function fall into this category. For fungi, most resembling the established borate transporter of brewer’s yeast (see “Bor1p” in FIGURE 4/Brewer’s Yeast) than “Nitro”. For animals, most resembling the seasquirt protein that shares a common ancestor with the vertebrate boron transporter BTR1 (see “BOR-like” in FIGURE 4/Sea Squirt). Thus an assignment of BOR-like character is kingdom-specific. For example, a BOR-like transporter from

AtBOR5* AtBOR6 AtBOR7

Alga DS:2G

OsBOR4

AtBOR2* AtBOR1* OsBOR1*

Alga DS:7

AtBOR3* Moss Moss PpBOR1 PpBOR2

“BOR” Group I “Primitive” * Demonstrated borate transporters

Moss DS:13

FIGURE 5. Relatedness among plantal Slc4-like proteins. The unrooted phylogram displays the relatedness, at the level of protein sequence, among the transmembrane domains of the 16 plantal Slc4-like proteins shown in FIGURE 4. Proteins are identified by the name of parent organism and the common name or the divergence score (DS), of each transporter, as listed in FIGURE 4. Note how the BOR and/or BOR-like transporters fall into two groups. It is unknown if members of Group I versus Group II are functionally distinct, like members of animal Slc4 groups in FIGURES 3 AND 6. The phylogram was generated using ClustalW (183) and TreeView (704).

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porter (BOR) function and thus we have assigned them as being AEs, NCBTs, and BORs. However, the function of many of the products is currently unknown. The phylogenetic relationships between the proteins in these groups are shown in FIGURE 5 (plants) and FIGURE 6 (animals, i.e., metazoa). We are not showing dendrograms for the other major taxonomic divisions that contain identified Slc4-like genes because: 1) bacteria have only two such genes, 2) the only known Slc4 sequences from phytoplankton are fragments from an unknown number of distinct Slc4-like products that cannot be meaningfully grouped, 3) the only two known amoebal genomes each has only one such gene, and 4) fungal Slc4-like genes are all BOR-like and differences among them appear to reflect mainly species divergence. We have attributed presently uncharacterized Slc4-like transporters to one of four groups, according to their relatedness at the protein level (within their transmembrane domains) to selected reference proteins.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Urchin DS:10

Placozoa SD:9 & 16 Squid DS:16 Anemone DS:8

Ciona “AE-like” nc

e

Anemone DS:11

re

Fly DS:15

fe

Sponge DS:9

Re

C68177

ro

up

NBCSA

Urchin DS:16

G

Anemone DS:24 Urchin DS:16

Grou

Nematode DS:18

p Refe

rence

ABTS-3

Ciona “NCBT-like” Urchin DS:22

Tablet DS:17 u Gro

pR

efe

r

e enc

Urchin DS:21 Sp-NBC

Squid NDCBE Squid NBCe Fly NDAE Nematode NCBT

Nematode DS:14

ABTS-1

ABTS-2

Nematode DS:6G ABTS-4

Generic

0.1

FIGURE 6. Relatedness among invertebrate Slc4-like proteins. The unrooted phylogram displays the relatedness, at the level of protein sequence, among the transmembrane domains of the 23 invertebrate Slc4-like proteins shown in FIGURE 4. Proteins are identified by the name of parent organism and the common name or the divergence score (DS), of each transporter, as listed in FIGURE 4. Note how the transporters fall into four groups: 1) including AEs and AE-like transporters (red), 2) including NCBTs and NCBT-like transporters (blue), 3) including BORs and BOR-like transporters (green), and 4) a “Generic” outlier (black) that does not fall into any of the three previous groups. The phylogram was generated using ClustalW (183) and TreeView (704).

worms is really “Ciona-BOR-like,” and not particularly “Bor1p-like” or “AtBOR1-like.” Within a kingdom, BORlike transporters cluster with their BOR-like reference protein on an unrooted phylogenetic tree (FIGURES 5 AND 6) and in the majority of cases are more similar to NCBTs than AEs.

We chose the sea squirt (Ciona intestinalis) as our animal reference point for items 2– 4 in the list immediately above because the sea squirt is the most primordial animal with three genes, each of which, on the basis of deduced amino acid sequence and conserved exon boundaries, shares a sin-

Table 3. Relatedness among representative Slc4-like transporters from bacteria and eukaryotes Domain:

Bacteria

Kingdom:

Eukaryota Amoebozoa

Plantae

Genus:

Nitrococcus

Dictostelium

Arabidopsis

Saccharomyces

Fungi/Metazoa

Gene product: Nitrococcus “Nitro” Dictyostelium Arabidopsis AtBOR1 Saccharomyces Bor1p Ciona AE-like Ciona NCBT-like Ciona BOR-like

“Nitro” 100

“Dicty” 33 100

AtBOR1 31 29 100

Bor1p 25 26 33

AE-like 36 27 28

NCBT-like 35 28 27

BOR-like 32 36 26

100

25 100

27 41 100

22 27 28 100

Ciona

Percentage identities among the transmembrane domain sequences of Slc4-like proteins. Identities were computed by pairwise BLAST (951). Accession numbers are provided in Figure 4. Note that gaps in protein sequence alignments (represented in Figure 2) reduce the computed percentage identity between Slc4-like proteins from different genera. GenBank protein accession numbers are provided in Appendix II.

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Ciona “BOR-like”

MARK D. PARKER AND WALTER F. BORON gle common ancestor with the three mammalian AEs (Ciona AE-like), or the five mammalian NCBTs (Ciona NCBT-like), or the singleton mammalian BTR1 (Ciona BOR-like).

For amoebae, we lack an amoebozoan reference protein. Therefore, in this special case, we compared the Slc4-like protein from social amoeba Dictyostelium to all of our Slc4-like reference sequences. It is revealed to share most identity with the Ciona BOR-like transporter (36%) and “Nitro” (33%; see TABLE 3). Thus we assign it as BOR-like with a DS of 3G compared with “Nitro” (FIGURE 4/Social Amoeba).

1) Of the many hundreds of complete bacterial genome sequences presently available, only two, those of the marine bacterium Nitrococcus mobilis and the opportunistic pathogen Segniliparus rugosus, contain an Slc4-like gene.5 2) In a sampling of 34 fungal genomes (not shown), each includes between one and three Slc4-like genes (all BORlike). About one-third of these genomes (predominantly of the classes Eurotiomycetes and Sordariomycetes) contain more than one Slc4-like gene. 3) A number of overlapping and nonoverlapping Slc4-like sequence fragments have been identified in the genome of the phytoplankter Emilyiana huxleyi (621, 795). Analysis of these fragments suggests that they might be derived from more than two, and perhaps as many as four, Slc4-like genes. Fragments of sufficient length to be reliably analyzed appear to be technically BOR-like, although generic. 4) Only two amoebal genomes are known, each from a different species of Dictyostelium. Each genome includes one Slc4like gene, both are technically BOR-like, but generic. 5) Plant genomes, which predominantly encode BOR-like products, include between two and seven Slc4-like genes (FIGURE 4/Plants), the number of genes being greatest in more recently emerged clades. This trend likely reflects a gradual accumulation of Slc4-like paralogs, resulting from gene/genome duplication. None of these products is more AE-like or NCBT-like than BOR-like. Some plant strains have multiple copies of the same BOR gene. For example, the boron tolerant “Sahara” cultivar of barley may have four times as many copies of the BOR1 gene as the boronsensitive cultivar “Clipper” (927).

Although our analysis is limited by the availability of complete genome sequences for key organisms, we find that the

6) Animal genomes include 2 or more Slc4-like genes, and most vertebrate genomes include at least 10. The number of Slc4-like genes is not always greater in more recently emerged clades, demonstrating that some Slc4-like genes have been lost following the emergence of certain clades (e.g., the fruit fly has fewer Slc4-like genes than most other animals). Animal Slc4-like products predominantly fall into the three categories: AE-like, NCBT-like, and BOR-like (which are underrepresented). Some fish genomes include two similar copies of each Slc4 gene, reflecting a recent genome duplication event.

4 The DS of an Slc4-like transporter is the difference between 1) the percent identity (computed by a pairwise BLAST alignment at http://blast.ncbi.nlm.nih.gov; see Ref. 951) of each transporter with its most similar reference protein and 2) the averaged percent identity of the transporter with the other reference proteins to which it has been compared.

5 Many partial, unattributed “Nitro”-like DNA sequences are present in the environmental genome database, making it likely that many other Slc4-like transporters are encoded in the genomes of marine bacteria. For example, nucleotide accession numbers AACY01572225, AACY01408744, AACY01136643, and AACY01500593.

For phytoplantkon, we also lack a reference protein. Compared against all other reference proteins, the four fragmented Slc4-like sequences appear to be primitive with DS of 3–5G. 1. Copy number of Slc4-like genes in diverse genomes

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Because the assignments to the AE-like, NCBT-like, and BOR-like groups are not always clear cut, FIGURE 4 includes-for each accession number-a “divergence score” (DS)4 that is a quantitative index of the protein’s divergence from a hypothetical “generic state.” A perfectly generic transporter, one that bears no greater resemblance of any one of the reference proteins to which it is compared, has a DS of zero. In the example case of “NCBT-like” transporters, the maximum DS is 66 because our reference NCBTlike transporter from Ciona exhibits an average 34% identity (i.e., 100% - 34% ⫽ 66%) with our AE-like and BORlike reference genes (TABLE 3). These scores provide an index of how “AE-like,” “NCBT-like,” or “BOR-like” any particular transporter is. We note that one nematode transporter with a DS of 6, which is more like the Ciona AE-like gene-product than either the NCBT- or BOR-like products, does not group with its assigned reference proteins in the phylogenetic tree in FIGURE 6. Thus we have assigned it as generic (noted by a “G” following the “DS” in FIGURE 4). Based on this assessment, we have also marked with a “G” plant and yeast transporters that have a DS of 6 or lower, indicating their possible generic nature. Our bacterial reference protein “Nitro” is the most generic of all of the protein considered here (DS of 3) compared with animal reference proteins, as befits its primitive nature.

number of Slc4-like genes varies on a genome-to-genome basis. Notable findings are as follows.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

In the following paragraphs we discuss in further detail the divergence of present day AE-like, NCBT-like, and BORlike genes from a single common Slc4-like progenitor. The actions and roles of nonvertebrate Slc4-like products are discussed in section III. 2. Archetypal and bacterial Slc4-like genes and products

A curiosity is that “Nitro” has ⬃20% sequence identity at the amino-acid level with certain prokaryotic sulfate permeases6 that share common ancestry with the Slc26 family of vertebrate anion exchangers. Thus, archetypal Slc4-like and Slc26-like genes may have been preceded by a single common ancestral gene. Furthermore, the genome of the archaebacterium Methanococcus maripaludis includes a sequence (YP_001548276) predicted to encode a multi-spanning membrane protein that is equally similar to “Nitro” and the prokaryotic Slc26-like paralog BicA (p. 820). 3. Emergence of BOR-like genes and products in fungi The known Slc4-like products of fungi are all BOR-like. The best known of these is Bor1p, the sole Slc4-like gene-product from the brewer’s yeast Saccharomyces cerevisiae, encoded by the BOR1 gene (FIGURES 2B AND 4/Brewer’s Yeast). Similar, singleton Slc4-like genes are found in the genomes of many other model, commercial and pathogenic species of fungi. The genomes of yet other fungal species, representing nearly onethird of fungal species whose Slc4-like genes have been reported, contain multiple slc4-like genes. For example, Aspergillus niger (FIGURE 4/Black Mold) has three. As with the BOR-like transporters of plants, those from fungi are more similar to each other than to those of other kingdoms, suggesting divergence from a single fungal BOR-like ancestor, but not necessarily indicating a common function. We discuss the action and role of Bor1p below.

6

For example, ZP_02948162 from Clostridium butyricum 5521.

The known Slc4-like products of plants are all either BORlike or “Primitive.” Slc4-like transporters in the plant kingdom are represented in FIGURE 4 by the genomes of one species each of green alga, moss, a monocotyledonous flowering plant, and a dicotyledonous flowering plant. The relatedness at the amino acid level of these transporters is represented in FIGURE 5. The green alga Ostreococcus is a unicellular organism and one of the smallest known eukaryotes, having only a single mitochondrion and a single plastid (206). The Ostreococcus “lucimarinus” genome includes two Slc4-like genes that appear to have diverged from a common ancestor. Both Ostreococcus transporters retain most similarity to the bacterial ortholog7 “Nitro,” and we thus consider them “Primitive.” However, because their divergence scores are small, they are also “Generic” or nearly “Generic.” Representing the Slc4 complement of an early land colonizing plant, the moss Physcomitrella retains a single “Primitive” Slc4-like gene along with two BOR-like genes. The two BOR-like genes appear to have diverged in the bryophyte lineage from a single common archetypal BOR-like ancestor that probably also gave rise to the rice and thale cress BORs in the “higher” plant/tracheophyte lineage. Thus the presence of recognizable BOR-like genes in plants appears to be contemporary with land colonization. During the emergence of “higher plants,” the archetypal plantal BOR-like ancestor appears to have diverged many times. The first duplication of the BOR-like ancestral gene appears to postdate the emergence of moss, but predate the divergence of monocotyledons (such as rice) and eudicotyledons (such as thale cress). One copy of the archetype retained a high degree of similarity to the original ancestral protein and gave rise to the precursor of AtBOR1–3 as well as OsBOR1 (BOR Group I, FIGURE 5). The second copy of the archetype gave rise to the precursors of AtBOR4 –7 as well as OsBOR2– 4 (BOR Group II, FIGURE 5). We discuss the actions and roles of plantal BOR products below. 5. Emergence of AE-like, NCBT-like, and BOR-like genes and products in animals The emergence of animals was more or less accompanied by the duplication of an archetypal Slc4-like gene, the inclu7 Following the recommendations of Jensen (447), we use the term ortholog to distinguish gene/proteins with a common genetic ancestry from different species (e.g., human SLC4A4 versus mouse Slc4a4) and the term paralog to distinguish gene/proteins that diverged from each other following gene duplication (e.g., human SLC4A4 versus human SLC4A5 or human SLC4A4 versus mouse Slc4a5). These terms are intended to refer to phylogenetic rather than functional relatedness.

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We have tentatively dubbed “Nitro” the gene-product from Nitrococcus (FIGURES 2B AND 4/Bacteria). A comparison of overall protein sequence identity shared between the transmembrane domains of “Nitro” and sea squirt Slc4s shows that “Nitro” itself is almost equally similar to AEs, NCBTs, and BORs (TABLE 3). It is interesting to note that the cytosolic C terminus (Ct) of “Nitro” and the Ct the Slc4-like gene product from Segniliparus both contain a sequence “LDA[D/ E]E” that is similar to the proposed binding site, in the Ct of mammalian Slc4 proteins, for carbonic anhydrase (CA) II (1007). Also notable, although perhaps coincidental, is that the Ct of the Segniliparus Slc4-like transporter and the Ct of human BTR1 both terminate with the sequence [D/E]xRP, although the significance of that motif has not been described for either protein.

4. Emergence of BOR-like genes and products in true plants

MARK D. PARKER AND WALTER F. BORON sion of sequence that encodes a substantial amino-terminal domain, and the subsequent evolution of distinct AE-, NCBT-, and BOR-like genes in animals. The overrepresentation of BORs and BOR-like transporters in the genomes of plants, amoebozoa, and fungi is complemented by their comparative underrepresentation in the genomes of animals (FIGURE 4/Animals). Most animals retain multiple AElike and NCBT-like genes but only a single BOR-like gene, with two exceptions: the nematode worm has two BORlike paralogs and the fruit fly has none.

The most primordial, animal Slc4-like sequence that represents a complete cDNA is the AE-like transporter known as “NBCSA” (847) from a sponge (FIGURE 4/Sponge). Notably, these placozoan and sponge Slc4-like proteins are predicted to have in place two features that are absent from Slc4-like proteins of plants and fungi, but that are found in vertebrate Slc4s: 1) a large cytosolic Nt and 2) an extended third extracellular loop-between the fifth and sixth transmembrane spans (TMs), which includes cysteine residues and multiple, putative N-glycosylation sites (FIGURE 2A). In Slc4 evolution, the large Nt appears for the first time in animals (e.g., tablet animal in FIGURE 4).8 The origin of the Nt is unknown, but it is likely to be derived from a preexisting open-reading frame that became appended to the transporter gene. An Nt-precursor gene is not identifiable as an isolated entity in any presently available genome sequence, nor is an Slc4-independent function for the Ntprecursor protein suggested by sequence homology to other soluble proteins. However, some mammalian Slc4 genes express variant transcripts that encode an isolated Nt (see below), which may be a vestige of the original genetic independence the of Nt-encoding sequence. It is noteworthy that a region of the crystal structures of the Nt of AE1 and NBCe1 shares substantial structural homology with some EIIA proteins, which are components of bacterial phosphotransferase systems that can act as soluble regulators of certain K⫹ channels and sugar transporters (245, 552).9 Clues to the divergence of AE-like, NCBT-like, and BORlike transporters in animals are provided by the visual guides to protein identity shown in FIGURES 4 AND 6. In tablet animals, two AE-like proteins are already distinct

8 Note that the extended Nt of the slime mold Slc4-like transporter bears no significant sequence identity to the Nt of animal NCBTs and likely evolved independently. 9 Precomputed structural alignments between the Nt of AE1 and certain bacterial EIIA proteins can be accessed via http:// www.ncbi.nlm.nih.gov/Structure/vast/vastsrv.cgi?sdid⫽51159.

816

6. Emergence of AE, NCBT, and BOR activity in animals Borate transport is likely a primitive function of Slc4-like transporters as evidenced by the presence of Slc4-like products with borate transport function in plants, fungi, and animals. Bicarbonate transport function appears to be a more recent specialization. To date, the only nonvertebrate Slc4-like gene-product with demonstrated Na⫹-independent Cl-HCO3 exchange activity is the AeAE from mosquitos (747), an ortholog of the AE-like transporter from Drosophila (FIGURE 4/Fruit Fly). Thus AE activity presumably arose prior to the divergence of protostomes (e.g., flies) from deuterostomes (e.g., mammals). Many nonvertebrate NCBT-like products have demonstrated NCBT function. NCBT-like proteins that perform Na/HCO3 cotransport are common to both coelomates (e.g., humans) and pseudocoelomates (e.g., ABTS-1 from C. elegans, see below). Thus it seems likely that NCBT-like products had, at the latest, acquired the ability to perform Na⫹-coupled HCO3⫺ transport soon after the emergence of the Bilateria, over 900 million years ago (368).

C. Divergence of Vertebrate Slc4 Genes From an Early Chordate Slc4-like Gene As far as we can discern from the presently available genome data, mammals, and likely most extant vertebrates, have at least one copy of each of the five distinct, known NCBT paralogs: Slc4a4, Slc4a5, Slc4a7, Slc4a8, and Slc4a10 (FIGURE 3). To investigate the genetic origins of these five genes, we look again to our reference genome from the sea squirt Ciona intestinalis, the genome of which includes one known NCBT-like gene. Ciona is a primordial chordate and shares a single common ancestor with all vertebrates. Thus the singleton Ciona NCBT-like gene is likely very similar to the archetypal vertebrate NCBT. 1. Analysis of exon-exon boundaries We can make some inferences about the emergence of chordate and vertebrate Slc4s from their common ancestor by

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The most primordial, animal Slc4-like transporters presently identified may be in the “tablet animal” Trichoplax adhaerens (FIGURE 4/Tablet Animal). An early draft of the Trichoplax genome sequence indicates the presence of at least three Slc4-like genes; two AE-like and one BOR-like.

from a BOR-like transporter. In our analysis, the most primordial organism with at least one gene each that is distinctly AE-like, NCBT-like, and BOR-like is the sea anemone (FIGURE 4/Starlet Sea Anemone). However, because the partial sequence that we have assigned as NCBT-like is only ⬃100 amino acids long, our assignment may not accurately reflect the nature of the full-length gene-product. Nevertheless, the divergence of NCBT-like genes must have occurred no later than the appearance of the Bilateria because NCBT-like genes appear in Coelomates and Pseudocoelomates (FIGURE 4).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

analyzing the exon boundaries of Ciona and vertebrate paralogs. FIGURE 7 shows a representative region of an Slc4 protein, from presumptive TM7 to TM14, aligned against the mRNAs that encode this region for all ten human SLC4 genes and all three Ciona Slc4-like genes.10 In the horizontal bars that represent mRNAs, different colored blocks represent sequences that are encoded by different exons. The analysis in FIGURE 7 shows that human SLC4s can be grouped into four categories by virtue of their gene structure. Group 1 is composed of the genes that encode the human Na⫹-independent Cl-HCO3 exchangers, AE1, AE2, and AE3. Group 2 is composed of electroneutral NCBT genes NBCn1, NDCBE, and NBCn2. Group 3 is composed of the electrogenic NCBT genes NBCe1, NBCe2, and the functionally controversial gene AE4. Group 4 is composed solely of the BTR1 gene that encodes the putative borate transporter.

The number of shared and unique exon boundaries among groups provides an indication of their relatedness. For gene regions encoding TM7 to TM14, the AEs (group 1) and NCBTs (groups 2⫹3), which share a single exon boundary (FIGURE 7A), are more closely related to one another than to BTR1. Most closely related to the Ciona AE-like gene is group 1, which shares five exon boundaries (FIGURE 7, B, D, G, J, AND L) with the Ciona AE-like gene, indicating that the Ciona AE-like gene shares a single common ancestor with all vertebrate AEs.

10 FIGURE 7 is based on the sequence alignments of Ciona and human Slc4 genes in Appendix III.

Protein 7

8

9

10

11

13

E

14

mRNA AE1 Group 1

AE2 AE3 Ciona AE-like NBCn1

Group 2

NDCBE NBCn2 NBCe1 NBCe2

Group 3

‘AE4’ Ciona NCBT-like BTR1

Group 4

Ciona BOR-like A

B C

D E

F G H

I

J

K

L

FIGURE 7. Analysis of common and unique exon boundaries among human SLC4 and Ciona Slc4-like genes. The gray horizontal protein bar represents a region of a typical Slc4 protein, between putative TMs 7–14. White numbered boxes within the protein bar mark the positions of ␣-helical TMs, similar to the representations in FIGURE 2B. Aligned below the protein bar are the corresponding regions of mRNAs that encode the TMs 7–14 region for all 10 human SLC4 genes and all 3 Ciona Slc4-like genes. Each mRNA bar is divided into colored boxes that represent the individual exons that comprise each sequence: thus a change in color on the horizontal axis marks the position of an exon boundary. Exons that appear to have been derived by splitting of a larger ancestral exon are colored green in the human lineage and purple in the Ciona lineage. Common exon boundaries that are discussed in the text are labeled A–L. The position of the exon boundaries for each gene are provided by NCBI Evidence Viewer (839). The sequence alignment from which this analysis is derived is presented in Appendix III.

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Most closely related to the Ciona NCBT-like gene structure are groups 2 and 3, which share four exon boundaries (FIGURE 7, A, C, E, AND K) with the Ciona NCBT-like indicating that the Ciona NCBT-like shares a single common ancestor with all vertebrate NCBTs. Exon boundaries “F,” “H,” and “I” (FIGURE 7), none of which is found in the Ciona NCBT gene, mark the divergence of group 2 genes (containing only

MARK D. PARKER AND WALTER F. BORON exon boundary “H” and “I”) and group 3 genes (containing exon boundaries “F,” “H,” and “I”). It is not necessarily the case that the gain of exon boundaries “H” and “I” predate the gain of boundary “F,” as introns may also be lost during the course of evolution (53), but the simplest explanation is that the group 2 (electroneutral NCBTs) archetype structure arose earlier and is the parent of the group 3 (electrogenic NCBTs plus “AE4”) archetype structure. Finally, the most recent NCBT gene divergence created the individual members of groups 2 and 3. Most closely related to the Ciona BOR-like gene is group 4 (i.e., BTR1). BTR1 shares no exon boundaries with the AEs and NCBTs in groups 2– 4, but shares six exon boundaries with the Ciona BOR-like gene (FIGURE 7).

2. Analysis of deduced amino acid sequences A) EMERGENCE OF THE FIVE NCBTS.

Among vertebrates, the earliest indicators of NCBT divergence are 41 NCBT-like gene fragments in the draft genome sequence of the sea lamprey Petromyzon marinus. By comparing fragments that have overlapping sequence homology, we estimate that there are at least two and perhaps as many as three NBCe1/ NBCe2-like genes (predominantly NBCe1-like) and at least two NBCn1/NBCn2/NDCBE-like genes (predominantly NBCn1/NDCBE-like). Thus it seems that the split between electroneutral NCBT-like and electrogenic NCBTlike genes predates the divergence of lampreys and jawed vertebrates. Although the fragmented and incomplete nature of the sequence information makes direct correlation of fragments to specific mammalian orthologs impossible, all five NCBT genes may already have been distinct entities by the time that lampreys appeared. The most primordial vertebrate NCBT cDNA sequence described may be a fragment cloned from a cartilaginous fish, the Atlantic stingray Dasyatis sabina (GenBank protein accession no. AAU29553). This cDNA fragment is most similar to mammalian Slc4a4 (NBCe1). The most primordial organism with a documented set of orthologs of the five mammalian NCBTs is the zebrafish Danio rerio, a bony fish. The zebrafish genome contains orthologs of all Slc4 genes, with the exception of Slc4a9, indicating that the five NCBTs were distinct entities at the point at which a common ancestral organism diverged to give rise to 1) rayfinned fishes (i.e., most modern bony fish, including zebrafish) and 2) lobe-finned fishes and tetrapods. B) EMERGENCE OF DUPLICATE NCBT-LIKE GENES IN BONY FISHES.

All vertebrates likely have a full complement of five NCBT

818

Let us consider the NCBT complement of zebrafish. For clarity we will provisionally refer to duplicate Slc4 genes as Slc4aX.1 and Slc4aX.2. In the case of Slc4a4, the sole reported Slc4a4.1 product (aka zNBCe1a aka zNBCe1-B aka NBCe1.1) most resembles the mammalian NBCe1-B variant (see below for a discussion of NBCe1 variants) inasmuch as it includes Nt sequence similar to the auto inhibitory and IRBIT (IP3 receptor binding protein released with inositol 1,4,5 trisphosphate)-binding determinants of NBCe1-B/C and terminates with an NBCe1-A/B-like Ct. An analysis of the Slc4a4.1 gene suggests that it would be unable to produce an NBCe1-A-like or an NBCe1-D-like transcript (926): specifically, 5= extension of exon 4a (see FIGURE 17) would not append a sequence to zebrafish NBCe1 that has obvious sequence similarity with the autostimulatory domain of mammalian NBCe1-A and NBCe1-D. Although Slc4a4.1 does have the capacity to encode a NBCe1-C like variant, the corresponding transcript has not been isolated. However, the presence of Slc4a4.1 variant products that lacks splice cassette I sequence (599), i.e., an NBCe1-E-like sequence indicates that posttranscriptional processing of NBCe1 does occur in these fish. Thus Slc4a4.1 is demonstrated to encode NBCe1-B and NBCe1-E-like sequences, but also has the potential to encode an NBCe1-Clike sequence. The sole reported Slc4a4.2 product (aka zNBCe1b aka NBCe1.2) is a partial clone that includes an NBCe1-B/Clike Nt, but lacks Ct sequence. A predicted complete open reading frame terminates with an NBCe1-C-like Ct that includes a PDZ-domain binding sequence. If each of the duplicate Slc4a4 genes has permanently taken on the character of a specific mammalian-like Slc4a4 splice variant, the distribution of “NBCe1-B versus NBCe1-C” in zebrafish could be controlled at the transcriptional level rather than, as in mammals, at the posttranscriptional level. Presumably these duplicate NCBT-like genes could serve as genetic back up for each other, although preliminary studies that reveal distinct distribution patterns (561) suggest that each may have carved out its own physiological niche. Slc4a9. The unusual Slc4a9 gene-product was initially described as NBC5 due to its relatedness to NCBT sequences, but was subsequently redesignated as AE4 following reports that the rabbit and rat orthologs are capable of Cl-HCO3 exchange activity. Slc4a9 clearly shares a common genetic origin with electrogenic NCBTs (FIGURES 3 AND 7), yet an Slc4a9 gene is notably absent C) EMERGENCE OF

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To trace the divergence of individual genes within these groups, we must rely on assessments of overall protein sequence relatedness, such as those presented in TABLE 3, depicted in FIGURE 3, and discussed in the following section.

genes, as evidenced by the presence of Slc4a4, Slc4a5, Slc4a7, Slc4a8, and Slc4a10 in the genomes of zebrafish and African clawed frogs (i.e., Xenopus). However, the complement of Slc4 genes may vary between genera. For example, due to a whole-genome duplication, zebrafish and many other fishes have two copies of at least Slc4a1 (561), Slc4a2 (882), Slc4a4 (167, 561), Slc4a5, and Slc4a10 (1101).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

from the draft genome of Danio rerio. Indeed, no Slc4a9 genes or products have been reported from any non-tetrapodan species. These observations suggest that Slc4a9 is a tetrapod-specific gene.

tetrapodan Slc4a9. It is more likely that a later Slc4a4 gene duplication in the tetrapod lineage gave rise to the precursors of mammalian Slc4a4 and slc4a9. Such interspecific divergence between Slc4a9 sequences could result in Slc4a9 products from different animals having different function and distribution, a subject that we discuss later in this review. Therefore, it may be helpful to think of Slc4a9 products not as a singular entity but as a group of related proteins, the genes for which diverged following a “recent” Slc4a4 duplication.

The deduced amino-acid sequence of Slc4a9 orthologs is not as well conserved as those of its closest paralog, NBCe1. Human, rabbit, rat, and mouse orthologs of NBCe1 are 96 –99% identical to each other, whereas Slc4a9 orthologs only share 79 –91% identity among those same species. The Nt sequence of Slc4a9 orthologs are more divergent (70 – 89% identity) than their TMD sequence (85–93% identity). The greater divergence of Slc4a9 compared with NBCe1 may reflect a reduced selective pressure to retain electrogenic NCBT function.

III. NCBTs AND RELATIVES IN NONMAMMALS A. Bacteria

human SLC4A4 fowl zebrafish Slc4a4 Slc4a4.2

NBCe1

1. Cation-coupled bicarbonate transport in bacteria CO2 sequestration by photosynthetic cyanobacteria makes a significant contribution to the global carbon cycle (284). The efficiency of carbon fixing by cyanobacteria is enhanced by a CO2-concentrating mechanism, of which cation-coupled HCO3⫺ transport is a vital component (recently reviewed by Price in Ref. 759). In animals, all Na⫹-coupled HCO3⫺ transport performed by Slc4 proteins. However, none of the prokaryotic cation-coupled HCO3⫺ transporters identified to date are Slc4-like. In the freshwater cyanobacterium Synechococcus sp. strain PCC 7942, HCO3⫺ transport is a high-affinity, primary active process that is induced under CO2-limiting conditions (701). HCO3⫺ transport is effected by an ABC (ATP binding

mouse Slc4a4 frog Slc4a4

zebrafish Slc4a4.1

frog “Slc4a9”

fowl “Slc4a9”

zebrafish Slc4a5.2 zebrafish Slc4a5.1 mouse Slc4a5

NBCe2

frog Slc4a5

human SLC4A9

human SLC4A5 mouse Slc4a9

“AE4” 0.1

fowl Slc4a5

FIGURE 8. Divergence of NBCe1, NBCe2, and “AE4” in vertebrates. The unrooted phylogram displays the relatedness, at the level of protein sequence, among the transmembrane domains of NBCe1, NBCe2, and “AE4” for zebrafish (Danio rerio), frogs (Xenopus tropicalis), fowl (Gallus gallus), mice (Mus musculus), and humans. The phylogram was generated using ClustalW (183) and TreeView (704). The GenBank protein accession numbers for each transporter are provided in Appendix IV.

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A comparison of vertebrate NBCe1, NBCe2, and Slc4a9 deduced protein sequences (FIGURE 8) provides important information about the origin of Slc4a9. The fish Danio rerio has no Slc4a9 gene, but two copies of the Slc4a4 gene (Slc4a4.1 and Slc4a4.2). The amphibian Xenopus tropicalis has an AE4-like gene, but Xenopus “AE4” is actually more identical at the amino acid level to human NBCe1 (66% identity between TMDs) than to human “AE4” (63% in the TMD). In the fowl Gallus gallus, “AE4” shares an equal degree of identity with human NBCe1 and human “AE4” (66% between the TMDs). Only in the mammalian lineage has the Slc4a9 gene diverged sufficiently to be clearly distinguishable from Slc4a4. The divergence of Danio Slc4a4.1 and Slc4a4.2, likely contemporary with a wholegenome duplication event (40), postdates the divergence of ray-finned fishes and lobe-finned fishes/tetrapods. Thus it is unlikely that either Slc4a4.1 or Slc4a4.2 is an ortholog of

MARK D. PARKER AND WALTER F. BORON

In another freshwater photosynthetic cyanobacterium, Synechococcus sp. strain PCC 6803, deletion of the cmp gene cluster has little effect on HCO3⫺ transport (877). Another transporter called BicA, a paralog of the vertebrate Slc26 family of anion exchangers, has been suggested to be responsible for HCO3⫺ uptake by Synechocystis PCC6803 under normal conditions (760). Furthermore, in this strain, CO2 limitation induces expression of a Na⫹-dependent HCO3⫺ transporter, called SbtA, that appears to have no eukaryotic equivalent. It is not known whether SbtA cotransports Na⫹ with HCO3⫺ and if so, in what ratio. However, it has been suggested that the process is driven by an inwardly directed Na⫹ gradient established by an active Na⫹-extrusion pump (877). 2. Bacterial Slc4-like transporters To date, the only reported occurrences of Slc4 orthologs in identifiable prokaryotic genomes are singular examples from the marine nitrifying bacterium Nitrococcus mobilis (FIGURES 2B AND 4/Bacteria) and the pathogenic bacterium Segniliparus rugosus. At 513 amino acids in length, the Nitrococcus clone, that we have provisionally termed “Nitro,” is the most compact of all known Slc4-like transporters. “Nitro” lacks many of the extended extramembranous regions of its vertebrate SLC4 counterparts, but is predicted to retain their topology in the transmembrane domain (FIGURE 2B). The codon usage pattern of the “Nitro” gene is more similar to that of eukaryotes than of bacteria (e.g., E. coli). The function of “Nitro” has yet to be fully characterized, but when heterologously expressed in Xenopus oocytes, Nitro does not mediate detectable HCO3⫺ transport but does permit the electroneutral and Na⫹-independent influx of 36Cl (713). It is intriguing to speculate that its retention in a nitrifying bacterium, which imports toxic NO2⫺ and exports NO3⫺, may indicate a role for “Nitro” in NO2-NO3 exchange. This hypothesis is especially tempting

820

in light of the homology between NO3⫺ and HCO3⫺ transporters in cyanobacteria (701), the role of an Arabidopsis ClC ortholog as a H/NO3 cotransporter (222), and the penchant of mammalian AE2 for NO3⫺ as a nonphysiological substrate (401). A possible BOR-like action of the archetypal Slc4-like gene-product is suggested by a consideration of Slc4-like transporters encoded in the eukaryotic domain. Only Slc4-like products with borate transport function are present across the kingdoms of plants (e.g., AtBOR1), fungi (e.g., Bor1), and animals (e.g., BTR1). Moreover, among plants, amoebozoa, and fungi, no HCO3⫺ transporters are known.

B. Fungi In eukaryotic organisms of primordial origin, such as yeast, no Slc4-like proteins with NCBT function have yet been identified. The best characterized example of a fungal Slc4like gene is BOR1 (aka YNL275W), the singular example from the baker’s yeast Saccharomyces cerevisiae. Its product Bor1p is a 576-amino acid nonglycosylated transporter that is similar in predicted secondary structure to its bacterial ortholog “Nitro,” except that the predicted Nt and Ct are slightly longer (FIGURE 2B). In Saccharomyces, Bor1p is localized to the plasma membrane (941, 1098), where it functions as a boron, or borate, efflux pathway, allowing cells to survive in media containing high levels of boric acid (689, 941, 943). A phosphate transporter, Pho88p, has been identified as a partner of Bor1p in a split-ubiquitin-based yeast two-hybrid screen (646), suggesting that Bor1p may be part of a larger integral membrane protein complex. In light of the observations that Bor1p is 1) not downregulated under boron-limiting conditions (442), 2) not strongly upregulated by high borate levels (442), 3) not the sole candidate borate transporter in this organism (116, 476, 689), and 4) does not compensate for boron efflux defects in an ATR1-deletion strain (476),11 it has been suggested that Bor1p may have another as yet uncharacterized function (442). A report that an overexpressed Bor1p-GFP fusion protein is enriched in vacuolar preparations compared with total cell homogenate (229) has been cited as evidence that the transporter is primarily localized to an internal compartment. However, this report should be interpreted carefully for three reasons. First, it conflicts with the earlier work on endogenously expressed Bor1p, noted above, that supports the plasma membrane localization of Bor1p protein and action (689, 941, 943, 1098). Second, overexpression of GFP-tagged Bor1p could swamp the trafficking machinery and lead to aberrant protein localization. Third, this report

11 Atr1p is a member of the multidrug-resistant transporter protein family originally noted for its ability to confer aminotriazole tolerance (464).

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cassette) protein assembly, called BCT1, encoded by the products of cmpABCD gene cluster (701). Together these four components create a complex equivalent to a mammalian ABC-type transporter that, in mammals, would be encoded by a single gene. The components of this transport complex are highly similar to those of a nitrate/nitrite transporter assembly from the same species, encoded by the nitrate assimilation (nirA) operon (701). Within BCT1, CmpA is an extracellular membrane-anchored HCO3⫺ binding lipoprotein that confers high affinity to the transport process. CmpB is the membrane-multispanning HCO3⫺ permease. CmpC and CmpD are intracellular ATPase subunits (511). CmpD is also predicted to have a solute-binding/transport modulatory role, based on its homology to the nitrite/nitrate transporter component NrtD (501). Structural data indicate that HCO3⫺ binding to the extracellular subunit CmpA is strongly Ca2⫹, but not Na⫹ dependent, although it is presently unclear whether Ca2⫹ is cotransported with HCO3⫺ (511).

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

also describes increased vacuolar fragmentation in a Bor1p deletant strain (229), which conflicts with the findings of a later study (442), thereby weakening the association between Bor1p and the vacuole.

C. Phytoplankton The phytoplankter Emilyiana huxleyi is surrounded by a shell (a coccosphere) composed of CaCO3 plates (coccoliths). Coccoliths are formed from Ca2⫹ and CO32⫺/HCO3⫺ in internal compartments (coccolith vesicles) and are exocytosed onto the cell surface. Coccoliths are an important sink of carbon in the global carbon cycle (e.g., the White Cliffs of Dover are composed of coccoliths), but the physiological role of coccoliths is unknown. One possibility is that coccoliths are a store of carbon for photosynthesis (860). Alternatively, coccoliths may be a sink for excess Ca2⫹, a desalting mechanism that would parallel the deposition of CaCO3 in the intestines of marine fishes. The molecular action of Slc4-like transporters in phytoplankton is unknown, but the abundance of one of these Slc4-like products in Emilyiana huxleyi increases in the presence of extracellular Ca2⫹. One possibility is that Slc4-like products might be responsible for HCO3⫺ influx across the plasma membrane (621). Another is that cytoplasmic carbonic anhydrases produce HCO3⫺ from CO2 when CO2 is abundant. In either situation, a transporter, conceivably an Slc4-like protein, would move HCO3⫺ from the cytoplasm, across the vesicle membrane, and into coccolith vesicles. However, the molecular actions and the subcellular locations of any Slc4like gene-product from phytoplankton are presently unknown. Considering the primordial boron transport function of Slc4-like proteins, it is also possible that some phytoplank-

D. Amoebae Valproic acid (VPA; 2-propylpentanoic acid) is a commonly prescribed anticonvulsant that acts upon ion channels as well as intracellular targets such as histone deacetylases (173). At doses above 1 mM, VPA is toxic to the model unicellular slime mold Dictyostelium discoideum; VPA-resistant strains have their singleton Slc4-like gene disrupted (960). The link between the Slc4-like gene and VPA transport in slime mold is strengthened by the inhibition of VPA uptake by the Slc4-blockers DIDS and tenidap and by inhibition of VPA uptake by extracellular HCO3⫺ (960). VPA uptake is independent of extracellular Na⫹ but stimulated by acidic extracellular pH (960). Thus it is possible that the protonated form of VPA moves into the slime mold, perhaps via an Slc4-like protein. Heterologous expression studies would be helpful to determine whether the Slc4-like protein is capable of such activity. It is unlikely that VPA is the physiological substrate of this transporter, and it is unknown if HCO3⫺ is carried by the Slc4-like transporter. An intriguing possibility is that VPA is a substrate or inhibitor of mammalian Slc4s. If VPA is a substrate of mammalian Slc4s, these transporters could promote VPA action upon intracellular targets. On the other hand, if VPA blocks neuronal NCBTs, the resulting fall in pHi could dampen neuronal excitability, contributing to the anticonvulsive properties of the drug.

E. Plants Algae, moss, and both mono- and dicotyledonous flowering plants each have their own unique complement of Slc4-like genes (FIGURE 4/Plants) that bear more sequence similarity among themselves than to any of their animal orthologs. These plantal Slc4-like genes are structurally similar to their yeast homologs, having short Nt and Ct cytosolic domains, but appear to always include an extended extracellular loop between TMs 9 and 10 (FIGURE 2B). The only plantal Slc4like transporters characterized to date are those from flowering plants. The founder member BOR1 (see next section) is a boron-efflux transporter from the thale cress Arabidopsis thaliana (943) and shares many properties with the yeast Slc4-like protein Bor1p. Boron is a highly significant element for plants: boron cross-linked rhamnogalacturonan II dimers are important cell-wall components (reviewed in Ref. 692). Too little boron can cause reproductive and

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Bor1p binds to stilbene derivatives such as DIDS and SITS (inhibitors of mammalian HCO3⫺ transporter), but there is no indication that Bor1p can transport HCO3⫺. Moreover, reports differ as to whether Bor1p-mediated boron efflux is inhibited by the presence of NaHCO3 in the growth medium (442, 943). Furthermore, as Na⫹ and Cl⫺ accumulation in yeast is unaffected by genetic ablation or overexpression of Bor1p (441, 442), it seems unlikely that Bor1p shares any common substrates with mammalian NCBTs. However, other anions may at least interact with Bor1p, as evidenced by the displacement of Bor1p from a SITS-affinity column by high concentrations of Br⫺, Cl⫺, HCO3⫺, I⫺, or NO3⫺ (1098). Borate efflux by yeast is faster at more acidic extracellular pH, which is consistent with the hypothesis that uphill borate efflux is driven by an inward H⫹ gradient (442), i.e., H/borate exchange. Inasmuch as the genetic diversity among Slc4-like products in fungi is at least as great as among their animal paralogs (e.g., human AE1 versus BTR1), it is possible that not all fungal Slc4-like transporters share the same molecular action as Saccharomyces Bor1p.

ton Slc4-like transporters might transport boron. Indeed, coccoliths do contain boron. However, the influx of uncharged H2BO3 across phytoplankton membranes is thought sufficient to account for the observed coccolith boron content (911). Even if this hypothesis were true, it would not preclude a role of an Slc4-like transporter as a borate efflux pathway in the plasma membrane, analogous to the role of Bor1p in yeast.

MARK D. PARKER AND WALTER F. BORON growth problems in plants, whereas excessive boron can be toxic (reviewed in Ref. 86). Boron transport is likely a complex process, aside from Slc4-like transporters, other plant proteins such as the aquaporin-like NIP5 (945, 1089) and NIP6 (947) are necessary for efficient boron transport throughout the plant (FIGURE 9).

and to seven BOR genes in thale cress (dicotyledonous), most paralogs within each group arose independently and the numbering is arbitrary. For example, although OsBOR1 in rice and AtBOR1 in thale cress are truly orthologous, OsBOR2 in rice is not the direct ortholog of AtBOR2 in thale cress. The relatedness of some plantal Slc4 products is as shown in the dendrogram in FIGURE 5.

For at least two reasons, the nomenclature for plantal Slc4like transporters requires careful interpretation: 1) Not all products named “BOR” have demonstrated boron transport function. 2) BOR genes from dicotyledonous genomes do not have exact orthologs in monocotyledonous genomes. Thus, although a common ancestor presumably gave rise to four BOR genes in rice (monocotyledonous)

B OsBOR1

AtBOR1/2

B (out)

NIP?

AtBOR4 NIP5

B (in)

Stele

EN (CS)

Cortex

EX (CS)

B (in)

EP

Stele

EN (CS)

Cortex

Rice root Cross section (elongation zone)

Arabidopsis root Cross section (elongation zone)

FIGURE 9. Roles of Slc4-like boron transporters in boron uptake and boron tolerance in the roots of plants. Radial cross sections through the roots of rice (A) and thale cress (B). Boron can enter the roots from the soil via the intercellular apoplast (blue shaded area), but is blocked from the root vasculature (in the stele) by casparian strips (CS). NIP boron channels, BOR1, and BOR2 transporters in root cells, provide a transcellular pathway through the epidermis (EP), exodermis (EX), cortex, and endodermis (EN) directing boron towards the stele and past the CS allowing boron to access the xylem. In thale cress, AtBOR4 directs excess boron into the soil.

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A

It is interesting to note that the plantal BOR-like transporters can be separated, according to protein sequence relatedness, into three distinct groups. The first group, “Primitive,” includes plantal Slc4-like products of unknown function. The second and third groups, “BOR group 1” and “BOR group II,” both include demonstrated borate trans-

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

As boron availability and toxicity are critical determinants of crop growth, and boron in plants acts as an antimicrobial agent, the function of BOR transporters and the linkage of BOR gene variation to enhanced boron tolerance is currently of considerable interest in plant physiology. Of special importance to animal physiologists is the growing number of reports of BOR-gene mutations, which may reveal much about the structure/function relationships of Slc4s. Notable is the coincidence, discussed below, that a mutation identified in the Arabidopsis AtBOR1 gene also occurs in human AE1, where the mutation is associated with hereditary spherocytosis. In the following two sections, we summarize the current knowledge concerning the physiological roles of BOR transporters in monocotyledonous and dicotyledonous plants. 1. Boron transport in monocotyledonous plants A) RICE.

The genome of rice (Oryza sativa) contains four Slc4-like genes named OsBOR1– 4 (FIGURE 4/Rice). OsBOR1 and OsBOR3 both mediate boron efflux. At present very little is known about the physiological roles of OsBOR2 and OsBOR4.

I) OsBOR1. The heterologously expressed OsBOR1-GFP fusion protein localizes at/near the plasma membrane of onion epidermal cells (674). In terms of the amino acid sequence, of the seven Arabidopsis AtBORs, OsBOR1 is most, and equally, similar to AtBOR1 and AtBOR2 (FIGURE 5). In terms of function, OsBOR1 is also similar to AtBOR1 and -2, mediating boron efflux at the level of individual cells, and mediating boron uptake (i.e., root to shoot) at the level of the whole plant (FIGURE 9A). Solutes and water can travel freely throughout the root apoplast (an extracellular space that includes cell walls) but are barred from the xylem-surrounding apoplast by two corky casparian strips. By analogy with boron uptake pathways in Arabidopsis, it is likely that boron crosses the basal membrane of rice root cells via aquaporin-like NIP5 transporters and

exits root cells across the apical membrane via BORs (256, 674, 944).12 The presence of OsBOR1 in root cells that span the exodermal casparian strip likely provides a transcellular efflux pathway by which boron is directed towards the endodermis where OsBOR1 in endodermal root cells would finally transport boron into the xylem-surrounding apoplast of the stele on the other side of the endodermal strip (674). In the root cells surrounding the xylem, the expression of OsBOR1 is constitutive under normal-boron conditions and is only modestly increased by boron starvation (674). On the other hand, in the exodermis, prolonged boron deficiency massively increases the expression of OsBOR1 (674), thereby enhancing boron extraction from the soil. II) OsBOR3. OsBOR3 functions as a boron-efflux transporter necessary for normal growth under boron-limited conditions (675) and probably plays a similar role to OsBOR1. The expression of OsBOR3 is regulated such that the OsBOR3 promoter drives exodermal expression in root tips, but endodermal expression in the root elongation zone (675). B) BARLEY. Variations in the sensitivity of barley cultivars to high boron levels have been linked to the Slc4-like HvBOR2 (aka bot1) gene locus (788, 927). Of the transporters displayed in FIGURE 5, HvBOR2 is most like OsBOR2. HvBOR2 transcripts are expressed in roots and leaf blade tips. In the latter, the transporter may contribute to the excretion of boron in guttation fluid (927), the liquid that some vascular plants secrete onto the leaf surface.

Four observations underlie the boron-tolerance of the hardy “Sahara” cultivar, which can withstand high boron levels, compared with boron-sensitive cultivars such as “Clipper” and “Schooner”: 1) Southern blotting suggests that the genome of “Sahara” may have four times as many copies of the HvBOR2 gene than “Clipper” (927). 2) Realtime quantitative PCR (qPCR) suggests that HvBOR2 transcripts may be many hundred-fold more plentiful in “Sahara” than in “Clipper” and “Schooner” (788, 927). 3) In conditions of elevated boron levels, “Sahara” maintains an abundance of HvBOR2 transcripts, whereas “Schooner” is unable to substantially increase HvBOR2 transcript abundance from its constitutively low level (788). 4) Heterologously expressed “Sahara” HvBOR2 is superior to “Clipper” HvBOR2 at enhancing the boron tolerance of yeast (927). Of 11 differences in nucleotide sequence between the 2 cDNAs, 2 are predicted to change the protein sequence. The first is the “Clipper” L305S polymorphism, which would disrupt a conserved Leu residue in putative TM8.

12 According to the terminology recommended in Reference 296, apical membranes face the shoot apex and basal membranes face the root apex.

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porters (marked with an asterisk in FIGURE 5, and discussed in the following sections). By analogy to similar groupings observed for mammalian Slc4s (e.g., FIGURE 3), it is possible that the molecular action of borate transport is different between groups I and II. However, nothing is presently known about the borate transport mode of any plantal transporter. The sole feature by which BOR-like transporters currently can be categorized is the ability to confer tolerance to low-boron stress versus high-boron stress. This distinction may follow the polarity of BOR expression within plants cells: a boron-efflux transporter in the apical membrane of plant cells will move boron in the direction of the shoots, whereas a boron-efflux transporter in the basal membrane will move boron in the direction of the soil (FIGURE 9). Note that both groups I and II contain members that confer tolerance to low-boron stress (e.g., AtBOR1 and OsBOR3).

MARK D. PARKER AND WALTER F. BORON The orthologous mutant L750C in rat NBCe1 causes a 50% loss of wild-type activity and is predicted to be located in a very conformationally sensitive part of the ion-translocation pathway (633). Thus, if borate transport via HvBOR2 and bicarbonate transport via NBCe1 share commonality in their translocation pathways, one explanation for the relatively poor ability of the “Clipper” versus “Sahara” gene to confer boron tolerance to yeast is a disrupted boronefflux pathway. The second polymorphism is D592G, in a poorly conserved region of the cytosolic COOH terminus, close to the last putative transmembrane segment. What effect, if any, this amino-acid substitution would have on the HvBOR2 transporter has yet to be elucidated. C) WHEAT.

Arabidopsis thaliana is a popular model organism used for the study of flowering plants. The Arabidopsis genome includes seven Slc4-like genes (FIGURE 4/Thale cress and FIGURE 5).

With regard to AtBOR1 localization at the tissue level, AtBOR1-GFP is expressed throughout the roots, but only under boron-limiting conditions, and mainly in the apical membranes of cells (see footnote 12), opposing the basal distribution of the boron-uptake transporter NIP5 (944). AtBOR1 expression in the roots is also enriched in the membranes of endodermal (EN) cells surrounding the stele that contains xylem vessels (650), which transport water and solutes from the roots to the rest of the plant (FIGURE 9B).

I) AtBOR1. In 1997, Noguchi and co-workers (686) reported the creation, by tilling, of a mutant Arabidopsis strain called bor1–1. Unlike wild-type plants, bor1–1 failed to thrive in boron-limiting conditions and produced fewer seeds. Linkage analysis showed that the reduced boron content of this and a similar mutant cultivar is due to mutation of the AtBOR1 gene (943). This study provided the first evidence, in any species, of an Slc4-like gene-product being involved in boron transport. One of the mutations in AtBOR1, G86E, is located in the short intracelullar sequence linking putative TM segments 2 and 3 (943) (see FIGURE 2B for putative BOR1 topology) and is orthologous to the naturally occurring human AE1 mutation G455E, which is associated with hereditary spherocytosis (433). It is not yet clear for either AE1 or AtBOR1 whether the G to E mutation causes a functional or trafficking defect in the transporter. A second mutant Arabidopsis strain bor1–2 is associated with an S74P mutation in AtBOR1, at a position midway through putative TM2 (943). The inappropriate positioning of a Pro residue in a helical region is likely to be very disruptive. For example, the L522P mutation in TM4 of human NBCe1-A leads to rapid protein degradation (241).

The cartoon in FIGURE 9B outlines a proposal for how the thale cress root transports boron from the soil to the xylem (568, 789). Solutes and water can travel freely throughout the root apoplast (extracellular space) but are barred by a single casparian strip from the apoplast surrounding the xylem. Boron enters root cells via aquaporin-like NIP5 transporters on the basal membrane (256, 944). The presence of AtBOR1 in the apical membrane of root cells provides a transcellular efflux pathway that directs boron in stepwise fashion through the cortex towards the endodermis, where AtBOR1 would finally transport boron across the casparian strip into the xylem-surrounding apoplast of the stele. Indeed, bor1–1 mutants have reduced boron content in the xylem exudate (943) as well as in shoots, shoot apices, and rosette leaves (650), but not the roots (943). Thus the critical AtBOR1-dependent step in the “root to shoot” boron-transport pathway is presumably the endodermal step responsible for xylem loading (943). This hypothesis is consistent with the observation that AtBOR1 enhances plant tolerance to boron-limiting conditions, and also explains why, when AtBOR1-GFP is overexpressed in a transgenic Arabidopsis strain, the protein does not afford increased protection from toxic levels of boron (650). How-

2. Boron transport in dicotyledonous plants A) THALE CRESS.

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In wheat (Triticum aestivum), TaBOR2 is an OsBOR2- and bot1/HvBOR2-like gene-product associated with increased high-boron tolerance in certain cultivars (788), consistent with a role in root to soil boron efflux. The TaBOR2 gene is expressed at a higher level in the boron-tolerant “India” cultivar over the boron-sensitive “WIMMC*10” cultivar (788). Furthermore, in response to boron-excess, boron-tolerant strains maintain substantial TaBOR2 transcript levels, whereas boron-sensitive strains are unable to substantially upregulate TaBOR2 transcript abundance from its constitutively low level (788).

The evidence that AtBOR1 is a plasma-membrane protein is the localization of heterologously expressed AtBOR1-GFP fusion protein at/near the plasma membrane of tobacco leaf cells (943). Five lines of evidence indicate that AtBOR1 mediates boron uptake at the organismal level (i.e., boron efflux from root cell into the xylem; see FIGURE 9B) and makes an important contribution towards “root-to-shoot” boron transport. 1) The AtBOR1 gene locus is associated with tolerance to boron-limiting conditions (1089). 2) In a transgenic Arabidopsis strain, boron-limiting conditions upregulate the expression of AtBOR1-GFP in roots, whereas the protein is mainly expressed in shoots when boron is in ready supply (942). 3) The restoration of high levels of boron after a period of boron limitation triggers the endocytosis and degradation of AtBOR1-GFP (942). 4) bor1–1 mutant plants, with mutant AtBOR1 genes, have a reduced boron content (650, 943) and exhibit reduced dimerization of rhamnogalacturonan II (see above and Ref. 685). Conversely, 5) the overexpression of AtBOR1-GFP in transgenic plants enhances boron accumulation in the shoot and shoot apices by about five times (650).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

dopsis has no reported boron-related phenotype (653). AtBOR5 transcripts are enriched in guard cells and sepals (1038) and are upregulated approximately eightfold in response to nitrate starvation of seedlings (1118).

II) AtBOR2. This transporter shares many properties with AtBOR1, to which it is 90% identical. Like AtBOR1, AtBOR2 mediates boron efflux when heterologously expressed in yeast (652). Disruption of the AtBOR2 gene in Arabidopsis is associated, under boron-limiting conditions, with retardation of both overall plant growth and elongation of cells at the root tip. Indeed, the AtBOR2 promoter is active in root tips (652). In seeds, a 24-h imbibing period induces AtBOR2 gene expression (1038). AtBOR2 likely contributes, in parallel with AtBOR1, to the directed “rootto-shoot” movement of boron depicted in FIGURE 9B.

VI) AtBOR6 and -7. Very little information is available on the expression and function of AtBOR6 and AtBOR7. AtBOR6 transcripts are enriched in mature versus immature pollen (1118).

III) AtBOR3. Transcripts have been detected in shoot guard cells, trichomes, and root cortex (653) as well as stigma and ovaries (1038). Although AtBOR3 mediates boron efflux when expressed in yeast, disruption of the AtBOR3 gene in Arabidopsis has no obvious phenotype. However, a bor1/bor2/bor3 triple mutant suffers more root-growth retardation than a bor1/bor2 double mutant, consistent with the hypothesis that AtBOR3 activity can compensate for defects in AtBOR1 and AtBOR2 expression (653). Thus AtBOR3 likely contributes to the directed “root-to-shoot” movement of boron depicted in FIGURE 9B. IV) AtBOR4. The distribution of a GFP-tagged AtBOR4 construct in Arabidopsis plants indicates that this transporter is normally expressed in the basal (soil facing) membranes of root epidermal cells (651) and transcripts are additionally detected in stamens (1038). In the root cells of transgenic plants, elevated boron levels result in increased AtBOR4-GFP expression (651). Furthermore, transgenic Arabidopsis and Sativa (rice) that are overexpressing AtBOR4-GFP, from a non-native promoter, exhibit enhanced tolerance to boric acid (461, 651). These observations are consistent with the idea that AtBOR4 normally exports excess boron from root to soil (651), as depicted in FIGURE 9B. An unexpected observation is that transgenic rice plants that heterologously express exceptionally large quantities of AtBOR4-GFP RNA (⬃100-fold more than the aforementioned transgenic rice plants tolerant to boric acid) exhibit a paradoxical diminution in tolerance to boric acid (461), as if excessive expression of AtBOR4 creates a rootto-shoot boron transport pathway. In principle, this could result from errant accumulation of AtBOR4 in the apical membranes of root epidermal cells, where AtBOR4 activity would parallel that of AtBOR1 and AtBOR2. V) AtBOR5. AtBOR5 mediates boron efflux when expressed in yeast (653). The AtBOR5 gene is situated in a genetic locus associated with tolerance to low-boron stress (1089), although disruption of the AtBOR5 gene in Arabi-

B) GRAPEVINE. Analysis of the Vitis vinifera genome indicates that grapevine plants have six Slc4-like genes, all of which are BOR-like (735).13 Three are most similar to AtBOR1 and -2 and are members of group 1 in FIGURE 5. Three are most similar to AtBOR4 and -5 and are members of group II in FIGURE 5. Interestingly, none of the six Vitis BOR-like genes appear to be direct orthologs of any of the seven Arabidopsis BORs (735). The lack of orthology between Arabidopsis and Vitis BORs is likely a complication of independent gene-duplication and gene-loss events following the divergence of the two organisms from a common ancestor.

I) VvBOR1. Although not a direct ortholog of AtBOR1, VvBOR1 is at least the most closely related paralog of AtBOR1 in the grapevine genome. Heterologous expression of VvBOR1 compensates for loss of boron efflux pathways in Bor1p-deficient Saccharomyces as well as in AtBOR1deficient Arabidopsis (735), indicating that VvBOR1 is a boron efflux transporter and normally plays a role in “rootto-shoot” boron transport (mimicking the action of AtBOR1 in FIGURE 9B). It has been suggested, in light of the reduced fertility of bor1–1 mutant plants, that VvBOR1 action promotes the fertility of individual Vitis flowers and thereby reduces the incidence of formation of small, seedless “shot” berries that can be formed parthenocarpically from unfertilized flowers (735). Consistent with this hypothesis, VvBOR1 transcript abundance and boron content are both lower in shot versus seeded grapes, although a causal link has yet to be established (735).

F. Invertebrate Animals The first NCBT to be described, the Na⫹-driven Cl-HCO3 exchanger, was originally detected in squid axons and snail neurons (105, 106, 111, 826). Moreover, experiments on these preparations, as well as barnacle muscle fibers (110) and crayfish neurons (658), first elucidated the importance of these transporter activities for pHi regulation. In the post-genomic era, many invertebrate HCO3⫺ transport activities have been specifically attributed to Slc4-like products. It is possible to see structural features, actions, and physiological roles of these transporters that are shared

13

The GenBank accession numbers are provided in Appendix II.

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ever, in the context of a unicellular organism, heterologous expression of AtBOR1-GFP in a Bor1p-deficient yeast strain promotes boron efflux and increases tolerance to boric acid (650, 943).

MARK D. PARKER AND WALTER F. BORON with some mammalian Slc4s. The invertebrate Slc4-like proteins are structurally diverse, but have a similar inferred topology to their vertebrate counterparts, including extended Nt and Ct (e.g., FIGURE 2A).

trans-side requirement for Cl⫺ to mediate Na⫹ and HCO3⫺ efflux) have not been formally demonstrated. At least the molecular action of ABTS-1 is qualitatively indistinguishable from that of the Na⫹-driven anion exchanger NDAE1 from Drosophila (see Ref. 71).

1. Sponge

2. Nematode worms The C. elegans genome contains four Slc4-like genes, abts-1 (anion bicarbonate transporter-1 aka CeNBC) through abts-4 (FIGURE 4/Pseuodocoelomata). The abts-1 gene has two alternative promoters that are active in different cell types (71), and abts-4 has at least two splice variants (876). Yet more predicted transcript variants are represented on Wormbase.14 A) DISTRIBUTION. In transgenic worms, the promoters of these four abts genes drive GFP expression in neurons (abt-1– 4), hypodermal cells (abts-1 and -3), body wall, pharynx, and vulval muscle cells (abts-1) as well as the intestine (abts-1 and -4) (71, 584, 876). Additional sites of expression for ABTS-2 and ABTS-4 protein are revealed in transgenic worms in which the natural termination codons of the genes are replaced by an in-frame GFP open-reading frame. In these animals, ABTS-2-GFP is expressed in the excretory cell of larvae and the ovaries of adults, whereas ABTS-4GFP is expressed in gut cells (876). Both transporter fusions exhibited a basolateral distribution. B) MOLECULAR ACTION.

To date, only the function of ABTS-1 (aka ceNBC), which, of the four ABTS proteins bears most sequence similarity to vertebrate Slc4s, has been characterized in detail. When heterologously expressed in Xenopus oocytes, ABTS-1 mediates a robust 36Cl influx and also mediates a detectable Cl-HCO3 exchange activity (71, 876). Furthermore ABTS-1 mediates electroneutral Na/HCO3 cotransport (71, 804). Taken together, these actions could be consistent with Na⫹-driven Cl-HCO3 exchange, although the hallmarks of classical NDCBE activity (i.e., Na⫹- and HCO3⫺-dependent Cl⫺ efflux and an absolute 14

http://wormbase.sanger.ac.uk.

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ABTS-1 also transports iodide (71). Of additional interest are the observations that abts-1 transcript and protein abundance doubled during arsenite exposure and that abts1-null worms are hypersensitive to arsenite toxicity (584). Arsenite causes intracellular acidification in a human cell line (425), leading Liao and co-workers to suggest that, if arsenite also lowers pHi in worms, abts-1-null worms may be unable to adequately counter the drop in pHi, leading to apoptosis (584). Although untested, another intriguing possibility is that ABTS-1 itself might counter arsenite toxicity by providing an arsenite-efflux pathway, paralleling the borate tolerance conferred by yeast and plantal Slc4-like products. The substrates of ABTS-2, -3, and -4 are unknown. ABTS-2 does not mediate substantial Cl⫺ or oxalate2⫺ uptake when expressed in Xenopus oocytes (876). C) PHYSIOLOGICAL ROLE OF ABTS-1.

In mammals, the concerted efforts of a Na⫹ driven Cl-HCO3 exchanger (NDCBE) and a K/Cl cotransporter (KCC-2) are hypothesized to play a role in nervous system maturation by lowering intracellular [Cl⫺] and potentiating the inhibitory effect of GABAergic and glycinergic signaling. In the case of C. elegans, ABTS-1 (together with KCC-2) is thought to play a similar role in the maturation of GABAergic signaling (see Refs. 71 and 948 as well as FIGURE 10) because of the following observations of neuronal hyperexcitability in abts-1-null worms. 1) abts-1-null worms are hypersensitive to the postsynaptic acetylcholinesterase inhibitor aldicarb as well as to the nicotinic acetylcholine receptor agonist levamisole (361, 584), indicating excessive ACh release. 2) Worms with a defective 5-HT reuptake transporter are typically hypersensitive to the inhibitory (i.e., hyperpolarizing) effects of 5-HT, causing the worms to slow down more than normal. Worms with mutations in the abts-1 gene (which would lead instead to depolarization) lose this hypersensitivity (361). 3) Hermaphrodite specific neurons (HSNs), which innervate the vulval muscles, express ABTS-1. In wild-type worms, the GABA receptor agonist muscimol inhibits egg laying via an inhibitory effect on the GABAergic HSNs. In worms carrying a mutation in abts-1, muscimol has no effect on egg laying, that is, GABA no longer elicits an inhibitory response. 4) Worms with a mutation in the G protein-coupled receptor EGL-47 (659) typically exhibit a reduction in egg laying.

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In the demosponge Suberites domuncula, cells associated with the siliceous endoskeleton contain transcripts encoding an Slc4-like transporter “NBCSA” (847). Three lines of evidence suggest that NBCSA may be a silicate transporter. 1) Many Slc4 transporters are stilbene-sensitive, and sponge cells have a DIDS-inhibitable silicate uptake activity; 2) NBCSA is the only Slc4-like transporter identified so far in this organism; and 3) NBCSA transcripts are upregulated in sponge cells by the presence of silicic acid in the bathing medium (847). No data address the issue of whether the transporter is Na⫹-coupled or has the ability to transport HCO3⫺.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

A

B

Neurons of wild-type worm

Neurons of abts-1 null worm

Agonist (e.g., GABA) K+

Agonist (e.g., GABA)

Cl–

Cl–

KCC-2

K+

ABTS-1

Na+

Cl–

KCC-2

2 HCO3–

Cl–

ABTS-1 BTS

Cl– –

KCC-2 and ABTS-1 lower [Cl–]i Vm more positive than ECl Stimulus is hyperpolarizing Neurotransmitter release inhibited

1) 2) 3) 4)

Reduced ability to lower [Cl–]i Vm more negative than ECl Stimulus is depolarizing Neurotransmitter release stimulated

FIGURE 10. Role of the Na⫹-driven anion exchanger ABTS-1 in the neurons of nematode worms. In wild-type C. elegans worms, ABTS-1 and the K/Cl cotransporter KCC-2 lower intracellular [Cl⫺] rendering GABAergic, glutamatergic, and 5-HT-ergic signals inhibitory to neurotransmitter release (A). In abts-1—null worms, the reduced ability to lower intracellular [Cl⫺] reduces the potency of inhibitory signals (B).

Egg laying is restored in worms with an additional mutation in abts-1 (71). 5) Muscimol causes body-wall muscles to hyperpolarize, resulting in an increase in body length. However, in worms with a disrupted abts-1 allele, muscimol causes a decrease in body length (71). 3. Annelid worms Although the molecular identity of an annelid NCBT has yet to be elucidated, multiple studies have demonstrated the presence of NCBT activity in these organisms. NCBT activity in annelidan cells was first demonstrated in 1985 by Schlue and Thomas, who studied the medicinal leech Hirudo medicinalis (841). In leech Retzius neurons, Schlue and Thomas showed that pHi recovery from an acid-load in the presence of CO2/HCO3⫺ is mediated by the dual action of an amiloride-sensitive Na-H exchanger (NHE) activity as well as a SITS-sensitive electroneutral Na/HCO3 cotransport activity that was proposed, although not demonstrated, to be due to a Na⫹-driven Cl-HCO3 exchanger (841). A third pHi regulatory mechanism was described in leech neuropile glial cells, a SITS-insensitive NCBT, the activity of which was accompanied by a small membrane hyperpolarization (235). This electrogenic glial transporter, suggested to be an electrogenic NBC operating with a Na⫹: HCO3⫺ stoichiometry of 1:2, was later demonstrated to be blocked by DIDS (236) and to readily perform electrogenic Li/HCO3 cotransport (671). The DIDS-sensitive current carried by the transporter has a reversal potential close to the resting potential of the glial membrane (671) and thus the net direction of HCO3⫺ transport mediated by the leech

glial NCBT may either be inwards or outwards, depending on the membrane potential (Vm) of the cell (239) and the extracellular pH (233). In this way, HCO3⫺ transport across the glial membrane can substantially modulate pHo to counter changes induced by neuronal activity (234, 812). The high-affinity of the transporter (Km ⬍1 mM) for HCO3⫺ means that the system contributes to pHi regulation in these cells even in the nominal absence of HCO3⫺ (237). Further experiments on leech giant glia indicate that the action of electrogenic NCBT in these cells could influence the rate of glutamate uptake through excitatory amino acid transporters, contributing towards termination of synaptic transmission (238). In summary, functional data would suggest that the genome of the leech includes at least two NCBT-like genes. 4. Mollusks Snail neurons and squid giant axons are classic systems for the study of intracellular pH regulatory mechanisms, and it was in these cell types that the first Na⫹-driven Cl-HCO3 exchanger (105, 106, 111, 826, 965, 966) and K/HCO3 cotransporter (220, 386, 387, 1097) activities were identified. To date, three Slc4-like genes have been cloned from squid (i.e., Loligo pealei) giant fiber lobe: sqNBCe, sqNDCBE, and the AE-like “SF4” (FIGURE 4/Mollusca). It is unknown whether squid genomes include a BOR-like gene. Characterized as an electroneutral Na⫹-driven ClHCO3 exchanger, this gene-product is also known as “SF1” (1008). This is not an Slc4a8 gene product, but sqNDCBE at least shares a common ancestor with mammalian electroA) sqNDCBe.

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1) 2) 3) 4)

+

MARK D. PARKER AND WALTER F. BORON neutral NCBTs. Transcripts of sqNDCBE are detected by northern blot in the giant fiber lobe, optic lobe, heart, and stellate ganglion (1008). The physiological characteristics of sqNDCBE expressed in Xenopus oocytes differs in two respects from the Na⫹-driven Cl-HCO3 exchange activity reported from squid axons. 1) In oocytes but not axons, Li⫹ can support sqNDCBE-mediated HCO3⫺ transport ⬃75% as well as Na⫹. 2) In oocytes but not axons, sqNDCBE-mediated HCO3⫺ transport can be readily driven in the efflux direction by removal of bath Na⫹. The precise reasons for these discrepancies have yet to be resolved (1008).

B) sqNBCe.

The predicted amino acid sequence of sqNBCe (aka “SF3”) has a higher overall identity to the electroneutral rather than electrogenic vertebrate Slc4s. Thus it was surprising that SF3, when heterologously expressed in Xenopus oocytes, proved to be an electrogenic Na/HCO3 cotransporter, named sqNBCe, the first electrogenic NCBT to be cloned from an invertebrate (746). This observation highlights a potential problem with making sequence-based predictions of transporter function. sqNBCe transcripts have a very different distribution to those of sqNDCBE and are predominantly detected by Northern blot in the gill and heart with additional expression in the giant fiber lobe (746). As expressed in oocytes, sqNBCe is unable to support Li⫹-stimulated HCO3⫺ transport, unlike the situation for the electrogenic NBC from leech glia, studied in situ. An intriguing observation, again for sqNBCe expressed in oocytes, is that removal of extracellular Na⫹ causes a prolonged inhibition of the transporter that is not reversed by restoring Na⫹ to the bath (746). Its deduced amino acid sequence suggests that SF4, the third Slc4-like transporter to be cloned from giant fiber lobe, is an AE-like gene-product. The function of SF4 has yet to be reported.

Clues as to the role of NCBTs in mollusks come from a recent study of the cuttlefish Sepia officinalis. Adult cuttlefish counter acidosis under conditions of chronic hypercapnia by elevating plasma [HCO3⫺] (396). Cuttlefish express two NCBTs in their gill epithelia: “soNBC” and “soNDCBE” (362), orthologs of sqNBCe and sqNDCBE. If soNBC is an electrogenic NCBT, it would be positioned to mediate an increased branchial HCO3⫺ reabsorption in these animals under hypercapnic conditions, per the role of branchial NBCe1 in fish. However, soNBC transcript abundance is not specifically altered by chronic elevations of PCO2 in juveniles and is paradoxically decreased in embryos and hatchlings (362). It is possible in juveniles that upregulation of soNBCe occurs at the post transcriptional level, or that pH regulation is effected via an alternative mechanism. In snail neurons, an NDCBE-like activity contributes to pHi regulation (966) and therefore likely maintains neuronal excitability, per the action of NCBTs in mammalian neurons. However, it has not been formally established whether the activity described in snail neurons is mediated by a Na⫹-driven Cl-HCO3 exchanger or the tightly-coupled action of an AE and an NHE (966). 5. Insects No insect genome reported to date appears to contain more than three Slc4-like genes, and none is known to include a BOR-like gene. The best-studied insect genome, that of the fruit fly Drosophila melanogaster, encodes two Slc4-like proteins (FIGURE 4/Panarthropoda): NDAE1 and CG8177. Multiple splice variants have been reported for each geneproduct. Drosophila NDAE1 (“Na⫹-driven anion exchanger”) has been characterized as a Na⫹-driven ClHCO3 (or Na⫹-driven Cl-OH) exchanger with a small associated anion leak (71, 810). Drosophila NDAE1 is also capable of mediating a substantial DIDS-sensitive NO3⫺ influx when heterologously expressed in Xenopus oocytes (852). An ortholog of NDAE1, AgNDAE1, from the mosquito Anopheles gambiae, mediates a similar Na⫹-driven anion exchange activity and is also capable of mediating some I⫺ influx (589). A) NDAE1.

In Drosophila, NDAE1 is widely expressed throughout the gut, Malpighian tubules, nervous system, and sensilla, with the majority of protein being basolaterally distributed (589, 854). NDAE1 transcripts have also been demonstrated in a specific subset of myocytes15 in the heart region of the cardiac tube of Drosophila (737). In the mosquito Aedes ae-

C) SF4.

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15 Those that coexpress the cardiac homeotic products Tin and Abd-A. NDAE1 expression is dependent on the expression of Abd-A (737).

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In situ, squid-axon NDCBE activity requires ATP, possibly for the phosphorylation of the transporter or an essential activator (107, 221). In addition, three lines of kinetic evidence are consistent with the hypothesis that the squid-axon transporter, in situ, actually transports the NaCO3⫺ ion pair. 1) Reciprocal changes in [Na⫹]o and [HCO3⫺]o have no effect on the flux as long as the product [Na⫹]o ⫻ [HCO3⫺]o is maintained fixed at constant extracellular pH (pHo) (111). Indeed, at a fixed pHo, this product is proportional to [NaCO3⫺]o. 2) Changes in pHo have no effect on the flux as long as [NaCO3⫺]o is fixed (109). 3) The reversible stilbene derivative DNDS (a divalent anion) appears to be a competitive inhibitor not only with extracellular HCO3⫺ but also with extracellular Na⫹. A kinetic analysis is consistent with the hypothesis that DNDS in fact competes with the NaCO3⫺ ion pair (108). Studies in frogs (534) and rabbits (see p. 847) indicate that vertebrate NCBTs do not transport the NaCO3⫺ ion pair.

D) PHYSIOLOGICAL ROLE.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

gypti, multiple NDAE1 transcript splice variants have been detected in the Malpighian tubules of adults (1070), in which NDAE1 protein is localized to the basolateral membranes of principal cells (589). A preliminary immunohistochemical study also localized NDAE1 to the basolateral membrane of the anterior stomach epithelia of Aedes aegypti larvae (654). An ortholog of NDAE1 likely mediates the stilbene-sensitive Na⫹-driven Cl-HCO3 exchange activity that has been detected in locust neurons (851). Also like NDAE1, this locust transporter is active in the absence of HCO3⫺. The importance of NDAE1 for insect health is underscored by the lethal nature of a P-element insertion in the Drosophila ndae1 5= untranslated region (UTR) (810).

6. Echinoderms A single Slc4-like gene has been cloned from the testes of the sea urchin Strongylocentrotus purpuratus (358), although genome analysis suggests that sea urchins may have four other Slc4-like genes (FIGURE 4/Echinoderms). The NCBTlike protein product of the cloned gene-“Sp-NBC”-is concentrated in the flagellar membrane of sea urchin sperm, where, as suggested by the authors of that study, it may play

16 This terminology is not intended to infer that AaAE1 is a direct ortholog of mammalian Slc4a1, but instead refers to AaAE1 being the first of two Na⫹-independent AEs cloned from insects.

7. Urochordates The draft genome of the sea squirt Ciona intestinalis predicts the existence of three Slc4-like genes, one each that can be described as AE-like, NCBT-like, and BOR-like (FIGURE 4/ Chordata). An in situ hybridization study shows that the NCBT-like gene is transcribed in the brain and visceral ganglion of tailbud embryos (837). These data are reinforced by the distribution of Ciona expressed sequence tags (ESTs), which suggests an exclusively neuronal expression of the NBC-like gene (837). EST data further indicate that the AE-like gene is expressed in the digestive gland, heart, and hemocytes, whereas the BOR-like gene is expressed in the heart, hemocytes, and neural complex (837). At present, nothing is known of the molecular action or physiological role of the Ciona Slc4-like transporters.

G. Nonmammalian Vertebrates It is likely that most vertebrate genomes encode orthologs of the three AEs, five NCBTs, and the singular BOR encoded by the human genome. However, not all vertebrate genomes include an Slc4a9 ortholog. The following sections summarize our current knowledge of the five NCBTs, as well as the BOR, in nonmammalian vertebrates. Note that the overwhelming majority of the published work in this area concerns electrogenic NCBTs of fishes and amphibians. 1. Cartilaginous fishes As far as we are aware, only one study has addressed the role of NCBTs in cartilaginous fishes. From the spiny dogfish (Squalus acanthias), Bleich et al. (84) studied isolated perfused rectal gland tubules, which contribute to whole animal osmoregulation by secreting a hyperosmotic NaCl solution (137). The molecular mechanism of NaCl secretion by tubule cells is represented in FIGURE 11. In cells from these tubules, pHi recovery from an acid load (imposed by an NH4⫹ prepulse; Ref. 106) requires basolateral Na⫹, is slowed by removing CO2/HCO3⫺, and is inhibited by DIDS. Thus these cells probably have an NCBT at the basolateral membrane. In separate experiments, the authors also observed that either reducing basolateral [Cl⫺] or depolarizing the cell causes a slow rise in pHi. They proposed a basolateral Na⫹-driven Cl-HCO3 exchanger with an unexpected voltage dependence, which they explained by suggesting that depolarization leads to a rise in [Cl⫺]i, which in turn enhances Na⫹-driven Cl-HCO3 exchange. Other possibilities include the following: 1) basolateral Na⫹-driven Cl-HCO3 exchanger in parallel with a depolarization-induced alkalinization (DIA; see Refs. 884 and 885) that is totally independent of the HCO3⫺ transporter; 2) basolateral electroneutral NBC in parallel with

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B) CG8177 AND AeAE. CG8177 is an AE-like gene-product, the distribution and molecular action of which has yet to be fully elucidated in Drosophila. The name CG8177 refers to “computed gene.” However, a preliminary study suggests that the protein mediates a 36Cl influx when expressed in Xenopus oocytes (454). In Drosophila larvae, CG8177, also termed DAE (Drosophila anion exchanger), is located in the basal membranes of interstitial cells of the midgut (263). Although RNAi knockdown of CG8177 in the midgut did not result in a phenotypic change in one study, global knockdown of CG8177 is lethal (263). A study of one of the two CG8177 orthologs from the mosquito Aedes aegypti (termed AeAE or sometimes AaAE1),16 localized the transporter to the basal membrane of stellate cells in the Malpighian tubules (589, 747). The same distribution is also observed for the Anopheles gambiae ortholog AgAE1 (589). Besides the Malpighian tubules, “AE1” is abundant in the gastric cecae and anterior midgut of larval Aedes and Anopheles, the lumen of which maintains an extremely alkaline pH to aid digestion (589, 590). When heterologously expressed in Xenopus oocytes, AeAE mediates stilbene-sensitive, Na⫹-independent Cl-HCO3 exchange (747). AeAE could be responsible for the DIDS-sensitive basolateral ClHCO3 exchange activity detected in the anterior segment of mosquito rectal saltglands (912). Little is known about the second AE-like gene-product from mosquitos (AgAE2) except for a report that it is widely expressed thought the gut of mosquito larvae (589).

a role in sperm capacitation and regulation of sperm motility (358).

MARK D. PARKER AND WALTER F. BORON

Duct lumen

Tight junction

Interstitial fluid

Na+

K+ Na-K pump CFTR

Na+

–

Cl

Na+ ++

cAMP HCO3–

?

H+

NHE

Cl– H+

Na+ NKCC

–

2 Cl

HCO3–

CA

K+

KCNQ1

Cl–?

Slc4?

Na+

CO2 H2O

–

Rectal Gland

2 HCO3

Rectal gland epithelium Intestine

Rectum

FIGURE 11. Role of an NCBT in the rectal gland of cartilaginous fish. The rectal gland duct joins the intestine at a point upstream of the rectum (see cartoon dogfish). In rectal gland epithelia, the Na-K pump maintains a low intracellular [Na⫹] driving basolateral Na/K/Cl cotransporter (NKCC) activity that supplies Cl⫺ for secretion across the apical membrane by CFTR. Anion secretion draws Na⫹ and H2O through a paracellular pathway, resulting in the secretion of a NaCl-rich solution from the interstitial fluid/blood. CO2 accumulation is dissipated by intracellular carbonic anhydrase (CA). Respiratory acidosis is prevented by NHE and NCBT action. ⫺ NHE and NCBT could also support HCO3 secretion via the unidentified apical anion exchanger, that is likely a member of the Slc26 family (84). KCNQ1 is a voltage-sensitive K⫹ channel (1018), also known as Kv7.1.

a Cl-HCO3 exchanger, plus an independent DIA; and 3) basolateral electrogenic NBC in parallel with a basolateral Cl-HCO3 exchanger or Cl⫺ channel. The presence of a basolateral NCBT is consistent with the stimulation of rectal-gland NaCl secretion by the infusion of NaHCO3 into the blood, a maneuver that mimics the post-prandial metabolic alkalosis known as the “alkaline tide” (1042). Note that, in mammals, it is typically basolateral NBCe1 and/or NBCn1 that supports fluid and salt secretion across epithelia by maintaining pHi and, under stimulated conditions, supplying HCO3⫺ for secretion (e.g., see below).

mitochondrion-rich (MR) pavement cells17 of the gills as well as other cells in the intestines. A) MOLECULAR ACTION OF BONY FISH NBCe1. Currently, of the NCBT orthologs expressed by bony fishes, only NBCe1 has been cloned and functionally characterized. NBCe1 clones from Osorezan dace (Tribolodon hakonensis; Ref. 382), pufferfish (Takifugu obscurus; Ref. 526), and zebrafish (Danio rerio; Ref. 926) all mediate electrogenic Na/HCO3 cotransport activity when expressed in Xenopus oocytes. In addition, pufferfish NBCe1 is capable of electrogenic Li/ HCO3 cotransport (167). The electrogenicity of gulf toadfish (Opsanus beta) NBCe1 has not been demonstrated, but

2. Bony fishes In bony fishes, NCBTs are vitally important to pHi and salt homeostasis, processes that are mainly associated with the

830

17 According to terminology of Perry et al. (741), these cells are referred to as MR cells in freshwater fish and chloride cells in saltwater fish.

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K+

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

the clone mediates a HCO3⫺-dependent Na-influx into Xenopus oocytes with a Km for HCO3⫺ of ⬃8.5 mM (952).

Many of these observations could be explained if the HCO3⫺-independent conductance associated with pufferfish NBCe1 (observation 2 above) persists in the presence of CO2/HCO3⫺ and makes a substantial contribution towards Vm. Such a conductance could interfere with measurements of true Na/HCO3 cotransport activity. A similar phenomenon has been described in the case of the HCO3⫺-independent conductance associated with the human NBCe1 mutant A799V (721). The HCO3⫺-independent conductance associated with pufferfish NBCe1 is also reminiscent of the conductive features of trout AE1 (663) and mammalian NBCn1 (189). B) DISTRIBUTION OF NCBTs IN BONY FISHES.

In the gill lamellae of Osorezan dace, trout (Oncorhynchus mykiss), and zebrafish, immunocytochemistry confirms the basolateral distribution of NBCe1 protein in a subpopulation of MR cells (382, 726, 925). Here NBCe1 is located in a “cytoplasmic” compartment, which the authors of the dace study attribute to an extensive basolateral system of infoldings (382), similar to those of salamander and mammalian PTs (632). Indeed, these MR cells express many transporters common to mammalian renal epithelia (reviewed in Refs. 278, 406). In zebrafish gills, NBCe1 colocalizes with NCC in a subpopulation of MR cells that do not express AE1 or the H pump (561). Outside the gill, NBCe1 mRNAs are detected in trout heart, liver, stomach, white muscle (741, 742), toadfish brain (952) and zebrafish brain, intestine, skin, and eye (561), specifically in the corneal endothelium,18 ganglion cell layer, rods, and cones (925, 926). In zebrafish embryos, NBCe1 is localized to the pronephros, specifically the anterior tubules and ducts, optic cup, and the ependymal cells that line the brain ventricles (926). NBCn1 and BTR1 expression appears to be widespread in zebrafish, whereas transcripts of NBCe2, NDCBE, and NBCn2 appear partic-

18

aka the corneal posterior epithelium.

C) ROLE OF NCBTs IN BONY FISHES.

I) pH homeostasis. In primary cultures of gill epithelia from the freshwater rainbow trout, a stilbene-sensitive Na⫹-dependent HCO3⫺ transport process is a major contributor to pHi homeostasis (1043). A striking example of how NCBTs can contribute to the pHi homeostasis of teleost fish is provided by the Osorezan dace, which, aside from spawning season, lives in a lake that has a pH of ⬃3.5. However, the MR cells in the gills of Osorezan dace are uniquely able to adapt to the acidic environment by the coordinated transcriptional upregulation of NHE3, CA II, the Na-K pump, and NBCe1 (382), a complete branchial Na/HCO3 uptake system (FIGURE 12A) that closely resembles that responsible for HCO3⫺ reabsorption in the mammalian proximal tubule (PT). Other, unadaptable fish species cannot survive in these acidic conditions due to a fatal combination of acidosis and an inability to accumulate salts against the osmotic gradient. Indeed, in the gills of zebrafish, NBCe1 transcript abundance is reduced in response to water acidification (561). The molecular response of fishes to hypercapnia appears to vary among species, but generally hypercapnia results in a compensatory increase in HCO3⫺ reabsorption to counter acidosis. In trout, a hypercapnic challenge leads to a transient upregulation of branchial NBCe1 mRNA at 1– 4 h, and a delayed rise in renal NBCe1 mRNA levels from 6 to 24 h (741). In the marine eelpout (Zoarces viviparous), a 24-h exposure to hypercapnia causes a paradoxical decrease in NBCe1 mRNA abundance in the gills, whereas NBCe1 transcript abundance gradually increases during a 6-wk period of chronic hypercapnia (232). In eels, which reportedly have no branchial NBCe1, hypercapnia elicits a rapid upregulation of renal NBCe1 mRNA that can reach levels 300-fold greater than basal after 12 h (741). If these changes correlate with increased HCO3⫺ reabsorption by the gills and kidney, these would tend to protect the fish from acidosis. In the African lungfish (Protopterus annectens), on the other hand, the abundance of branchial and renal NBCe1, at least at the level of mRNA, are unaltered by acid-base disturbances. Instead, these animals use ventilatory control to blow off excess CO2 and thereby achieve whole body pH homeostasis (322). Regarding the liver, evidence suggests that an electrogenic Na/HCO3 cotransport activity is important for pHi homeostasis in trout hepatocytes (305), although, as in mammals (8), this activity may be attributable to NBCe2. Regarding the stomach, trout NBCe1 transcripts are detected in both the antrum and the corpus, where the trans-

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Pufferfish NBCe1, as expressed in Xenopus oocytes, exhibits a number of unique features not described for other NBCe1 orthologs: 1) oocytes expressing pufferfish NBCe1 are unusually loaded with Na⫹; 2) pufferfish NBCe1 exhibits an inwardly rectifying, HCO3⫺-independent, ion conductance; 3) pufferfish NBCe1 exhibits an inwardly rectifying CO2/HCO3⫺-dependent conductance; 4) the apparent Km of the cotransporter for extracellular Na⫹ is voltage dependent (the apparent affinity being lower in the negative voltage range); and 5) the reversal potential (Erev) for the cotransporter is not substantially altered by a reduction in [Na⫹]o, as if the stoichiometry of the transporter is variable (167).

ularly abundant in zebrafish brain and eye (561). Furthermore, in zebrafish, an abundance of NDCBE transcripts is notable in the heart and NBCn2 expression is notable in the spleen (561).

MARK D. PARKER AND WALTER F. BORON

A

B

Water

Tight junction

Interstitial fluid

Intestinal lumen

Interstitial fluid

3 Na+

ENaC H+

HCO3

2 K+

Na+

Na-K pump HCO3–

H+

Na-K pump HCO3–

CA

CO2 NBCe1

+

Na+

cAMP

3 HCO3–

H+

Gill MR Cell (Trout)

NBCe1

H2O Na+ 2 HCO3–

Intestinal Enterocyte (Pufferfish)

FIGURE 12. Role of NCBTs in the gills and intestines of bony fishes. In the gill epithelia of freshwater fish such ⫺ absorption from the water. In the gut epithelia of marine as trout (A), NBCe1 contributes to Na⫹ and HCO3 ⫺ fishes such as pufferfish (B), NBCe1 contributes to HCO3 secretion into the gut lumen. ENaC is an epithelial Na⫹ channel. The identities of the non-Slc4 transporters involved in these pathways have not been determined for all species in which these systems have been identified.

porter is hypothesized to support a protective secretion of HCO3⫺ onto the apical surface of the cells. However, contrary to those authors expectations, NBCe1 mRNA levels fell with dietary acidification (918). II) Salt homeostasis. Killifish (Fundus heteroclitis) are vulnerable to Na⫹ loss by fluctuation in salinity in their environment, as reviewed briefly by Scott et al. (856). In these fish (856) and in Japanese eels (981), NBCe1 is a constitutive player in a freshwater inducible branchial Na⫹ uptake system, similar to that shown in FIGURE 12A. Following the same theme, the MR cells of Mozambique tilapia (Oreochromis mossambicus) tend to exhibit reduced expression of NHE3 and NBCe1 when the salinity of their environment is increased, although the data do not achieve statistical significance (306). Marine fish desalt seawater as it passes along the gut. HCO3⫺ secretion into the gut lumen plays a key role in one aspect of this desalting, the removal of Ca2⫹ and Mg2⫹ from the gut lumen as the secreted HCO3⫺ precipitates concentrated divalent cations as an excretable carbonate deposit (reviewed in Ref. 1037). A role for NBCe1 in this process is suggested by the presence of NBCe1 transcripts in the intestines of the gulf toadfish (952), trout (343, 741), and pufferfish (526). Moreover, deposit

832

formation by isolated mucosal layers from the intestines of the sea bass (Cicentrarchus labrax) depends on basolateral Na⫹ and HCO3⫺ (281). Immunocytochemistry demonstrates a basolateral distribution for NBCe1 in intestinal epithelium (526), where the cotransporter would presumably operate with a 2:1 stoichiometry (FIG⫺ URE 12B). The apical step of HCO3 secretion is likely effected by Slc26a6 (526). Transferring these fishes from freshwater into seawater, or in the case of the toadfish from less saline to more saline seawater, causes the upregulation of NBCe1 transcripts in their intestine (343, 526, 952). This would presumably support the increased base excretion observed in these animals during seawater acclimatization (741). III) Development. Inhibition of NBCe1 translation in zebrafish embryos produces developmental defects, including hydrocephalus, retinal distention, and the presence of unidentified particulate matter in the ventricular spaces (926). 3. Amphibians The first description of a Na-coupled HCO3⫺ transporter that is independent of Cl, the electrogenic Na/HCO3 cotransporter, came from Boron and Boulpaep’s 1983

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CO2 H2O Na

2 K+

Slc26a6

CA

NHE3

3 Na+

Cl– –

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

study of pHi regulation in the proximal tubule of the tiger salamander Ambystoma tigrinum (103). They demonstrated NCBT activity at the basolateral membrane of the PT epithelia (103). A similar activity is present in the PT of the mudpuppy Necturus maculosus (606). The injection of poly(A) mRNA from salamander PT into Xenopus oocytes led to the cloning of the first NCBT cDNA, which encodes a protein termed aNBC (Ambystoma Na bicarbonate cotransporter; Ref. 809). We now recognize aNBC19 as Ambystoma NBCe1-A; a product of the Ambystoma Slc4a4 gene.20 A) MOLECULAR ACTION OF AMPHIBIAN NBCe1.

B) DISTRIBUTION OF NCBTs IN AMPHIBIANS. I) NBCe1. The majority of renal Ambystoma NBCe1 protein is localized to the basal folds of the late distal tubule of this mesonephric kidney, with a smaller basolateral presence in the PT (632, 844). A developmental expression pattern of NBCe1 in Xenopus laevis pronephroi is revealed by in situ hybridization experiments (1102). Low abundance of Xenopus NBCe1 (“XNBC1”) mRNA occurs in the early and late PTs at developmental stage NF29,21 but a greater abundance of XNBC1 is present in the late distal segment by stage NF33 (1102), where its distribution overlaps with that of Ca2 (1103). A more recent model of the Xenopus pronephros, based on a large-scale in situ hybridization mapping of transcripts, localizes a significant population of NBCe1 transcripts to an early distal tubule region “DT(1)” that is homologous to the mammalian thick ascending limb (581, 779). Aside from the pronephric presence, NBCe1 transcripts are detected in the cranial ganglia, nasal pit, otic vesicle, somites, hatching gland, and cement gland of Xenopus embryos as well as in the bladder, brain, and small intestine of Ambystoma (809).

19

GenBank protein accession O13134. Nomenclature guidelines for Xenopus genes are provided at http://www.xenbase.org/gene/static/geneNomenclature.jsp. 21 About 1.5 days post-fertilization. “Nieuwkoop Faber” developmental stages are defined in Ref. 1, and the images that accompany the defintions are reproduced online at the Xenbase: Xenopus laevis and Xenopus tropicalis biology and genomics resource (http:// www.xenbase.org/anatomy/static/NF/NF-all.jsp). 20

III) NBCn1. Slc4a7 transcripts that encode the electroneutral Na/HCO3 cotransporter NBCn1 are detected by in situ hybridization mainly in the central nervous system of Xenopus embyros, with an additional presence in the PT, epidermis and external gills. Slc4a7 is expressed at an earlier developmental stage than slc4a10. IV) NDCBE. We are unaware of any reports concerning the distribution of Slc4a8 products in amphibians, although if it exhibits a similar expression pattern to its mammalian ortholog, we might expect Slc4a8 to be abundantly expressed in neurons. V) NBCn2. Slc4a10 transcripts are detected by in situ hybridization mainly in the central nervous system (brain, retina, and spinal cord) and the pineal gland of Xenopus embryos and at a later developmental stage than Slc4a7. VI) Slc4a9. We are unaware of any reports concerning the distribution of Slc4a9 products in amphibians. VII) BTR1. Transcripts for another Slc4 family member Slc4a11 (referred to as “XNBC2”) are evident in the early PT at developmental stage NF26. Some transient expression also occurs in the early distal segment, but is much diminished by stage NF38 (1102). C) ROLE OF NCBTs IN AMPHIBIANS. I) Vision. In the amphibian eye,

electrogenic Na/HCO3 cotransport activity, presently unattributed to a specific NCBT, has been detected in 1) lens epithelia of the cane toad Bufo marinus (1039), 2) optic nerve glial cells of Necturus (50, 51), and 3) retinal glial cells of both Necturus (51) and Ambystoma (680). In retinal glial cells from Ambystoma, electrogenic NCBT activity is calculated to operate with a Na⫹:HCO3⫺ stoichiometry of 1:3 and is preferentially localized at the glial end-feet (681, 682). In glial cells from both the retina and optic nerve, the electrogenic NCBT activity contributes towards maintaining a slightly alkaline resting pHi (49, 681), implying that the electrogenic NBC mediates a net HCO3⫺ uptake even when operating with a stoichiometry of 1:3 (see below). When retinal glial cells from Ambystoma are depolarized, the resulting HCO3⫺ influx mediated by the electrogenic NCBT causes a drop in pHo that could serve to balance the extracellular alkalinizations resulting from neuronal activity or light stimulation of the retina (681). Because the NCBT-mediated drop in pHo is localized at

22 Data from The Xenopus Gene Expression Database (http:// www.euregene.org/xgebase/pages/entry_page.html).

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Currently, of the NCBT orthologs expressed by amphibians, only an NBCe1-A ortholog from the salamander Ambystoma has been cloned and functionally characterized. This protein is an electrogenic Na/HCO3 cotransporter with kinetic properties that are very similar to mammalian (i.e., rat) NBCe1-A (339). Like mammalian NBCe1, salamander NBCe1 is blocked by DIDS (339). Kinetic data suggest that an NBCe1/NBCe2-like activity in frog retinal pigment epithelium does not, unlike the NCBT activity in squid giant axons, transport the ion pair NaCO3⫺ inasmuch as the Km for Na⫹ of this activity appears to be independent of [CO32⫺] (534).

II) NBCe2. Slc4a5 transcripts that encode the second electrogenic NCBT, NBCe2, are detected by in situ hybridization of Xenopus oocytes and appear to persist in most tissues throughout embryonic development.22 Note that isolated, defollicated oocytes exhibit no detectable electrogenic NCBT activity (1009).

MARK D. PARKER AND WALTER F. BORON the glial endfeet that contact blood vessels, and because blood vessels dilate in response to a fall in pHo, it has been suggested that NCBT activity may also contribute to a mechanism that increases blood flow during neuronal activity (681).

A

II) Mucosal protection. In the stomach of the edible frog Rana esculenta, NCBT activity is present at the basolateral membranes of the oxynt(ic)opeptic cells that alternately secrete both HCl and HCO3⫺ at the surface of the gastric fundus (Ref. 209 and FIGURE 13B). In the edible frog, the NCBT activity is clearly mediated by an electrogenic Na/ HCO3 cotransporter, whereas in the in the North American bullfrog Rana castesbiana, it is not clear whether the transporter is electrogenic or electroneutral (1074). In both cases, NCBT activity, the basolateral step in the secretion of HCO3⫺ into the mucus that covers the stomach surface, would play a protective role by helping to counter the acidifying effect of HCl back-diffusion from the gastric lumen, and in the process would keep pHi relatively high (208, 1074). Electrogenic NCBT activity in the oxynt(ic)opeptic cells of Rana esculenta is stimulated by carbachol but inhibited by histamine (228).23

23

Although carbachol and histamine both stimulate HCl secretion.

B

Subretinal space

Tight junction

Choroid

Stomach lumen

Interstitial fluid

H2O Na-K pump

H-K pump 2 K+

K+

3 Na+

H+

3 Na+ 2 K+ Na-K pump

NKCC

Cl– HCO3–

Na+ AE

K+ 2 Cl–

Cl– HCO3–

NCBT

NCBT

Cl– Na+

HCO3–

2 HCO3–

2 HCO3–

Slc26?

Retinal Pigmented Epithelium (Frog)

Na+

Oxyntopeptic Cell (Frog)

FIGURE 13. Role of NCBTs in the retinas and stomachs of amphibia. In the retinal pigment epithelia of frogs (A), an electrogenic NCBT exhibits an unusual apical distribution and contributes towards fluid absorption from the subretinal space, promoting retinal attachment. Although undemonstrated in frogs, study of mammalian RPE indicates that the apical NCBT could be either NBCe1 or NBCe2 and the basolateral AE could be AE2 (11). ⫺ In gastric epithelia of frogs (B), an electrogenic NCBT contributes towards HCO3 secretion onto the cell surface ⫺ that protects cells from acid attack. The transporter responsible for moving HCO3 across the luminal membrane of these cells has not been identified, although both Slc26a6 and Slc26a9 have been suggested to perform this function in mammalian gastric mucosa (744, 1055).

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The apical membrane of bullfrog retinal pigment epithelia (RPE) has stilbene-sensitive electrogenic Na/HCO3 cotransport activity (400, 532) that is a major contributor to the transepithelial HCO3⫺ absorption from retina to blood. This HCO3⫺ absorption helps to drive fluid absorption across the RPE, preventing subretinal edema and promoting retinal attachment (533, 536). The absorption of HCO3⫺ per se lowers subretinal pHo (535). It has been suggested that the NBC activity may be responsible for the lowering of subretinal pHo that occurs in response to a light-induced reduction in [K⫹]o (585). FIGURE 13A shows a model of the role of electrogenic NCBT activity in the transepithelial ion transport across the amphibian RPE. In RPE, the apical polarity of normally basolaterally distributed transporters such as NCBTs and the Na pump is related to an unusual, partial reversal of polarized protein distribution in these cells (reviewed in Ref. 627). By analogy to the distribution of NCBTs in rats and humans, the apical NCBT in the RPE of bullfrogs is probably an Slc4a4 product (11, 94), although Slc4a5 transcripts are expressed in

human RPE and, in mice, retinal detachment is a phenotype of Slc4a5 gene disruption (see p. 880).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

In the Necturus PT, the movement of Cl⫺ across the basolateral membrane has a strong trans-side dependence on Na⫹ and HCO3⫺, consistent with the presence of a Na⫹driven Cl-HCO3 exchanger (356). On the other hand, the data are also consistent with the presence of a basolateral Cl⫺ channel in parallel with the subsequently identified electrogenic NBC activity (606), with voltage changes providing indirect coupling of Cl⫺ to Na⫹ and HCO3⫺. Viewed somewhat differently, if a cell has a Cl⫺ conductance and an

A Lumen

Tight junction

Interstitial fluid

NHE

Na-K pump 3 Na+

Na+ H+

++

cAMP

2 K+

electrogenic NBC in the same membrane, it would be very difficult, using only classical electrophysiological approaches, to resolve the presence of a Na⫹-driven Cl-HCO3 exchanger (see also our discussion of the dogfish NDCBE with unusual voltage dependence on p. 829). 4. Reptiles Orthologs of all mammalian Slc4s, with the exception of NBCe2, are identifiable in the draft genome of the green anole lizard Anolis carolinensis. However, we know of no definitive demonstration of NCBT activity in any reptilian cell or tissue. The lack of reports concerning reptilian NCBT activity is likely related to 1) the underrepresentation of reptiles among physiological model organisms and 2) the unusual acid-base physiology of the reptiles that have been studied. For example, Alligator mississippiensis excretes an unusually alkaline urine and has a low plasma [HCO3⫺] (566). The PT epithelia of these animals apparently do not express NHE or CAII (1000), proteins that are considered necessary for substantial HCO3⫺ reabsorption in mammals. Furthermore, the distal renal epithelia of alligators actually mediate a net secretion of HCO3⫺ under normal conditions (565, 1000), reminiscent of collecting ducts of mammals fed an alkaline diet (e.g., see Refs. 271 and 319). However, the working of an as-yet unidentified HCO3⫺ reabsorbtive mechanism is disclosed in alligator distal tubules when tubular HCO3⫺ secretion is blocked by acetazolamide (565). In alligators, HCO3⫺ secretion may serve to balance the renal excretion of NH4⫹ (due to the high pH of the urine, pNH3 in alligator urine is ⬃0.1 mmHg; Ref. 566) that is necessary due to their inability to synthesize urea (565). Even from studies of snakes, which are capable of acidifying their urine, there are no reports of NCBT activity in isolated proximal or distal renal tubules (217, 494). 5. Birds

NHE Na+

H+ HCO3–

cAMP

CA

++

H+

NBCe1-A Na+ CO2

3 HCO3–

H2O

Proximal or Distal Tubule Cell (Salamander) FIGURE 14. Role of NCBTs in the renal tubules of amphibia. In proximal and distal renal tubule epithelia of amphibia, NBCe1 con⫺ reabsorption pathway, tributes towards a H⫹ secretion/HCO3 which maintains whole body pH within a narrow physiological range.

Orthologs of all 10 mammalian Slc4s are identifiable in the draft genome of the fowl Gallus gallus. However, published studies of avian NCBT activity are few in number. Nephrons in avian kidneys are graded into three categories, according to differences in the length of their loops of Henle (briefly reviewed in Ref. 122): 1) long-looped “mammalianlike” nephrons; 2) short-looped “reptilian-like” nephrons; and, falling between the two extreme forms, 3) a population of “transitional” nephrons. A stilbene-sensitive NCBT activity, most consistent with the presence of NDCBE, is detected in isolated nonperfused PT from chicken transitional (493) and long-looped nephrons (122, 123), but not in short-looped nephrons (122, 629). NCBT activity has also been detected in studies of nonrenal avian cells. The steady-state pHi of cultured chick embryonic heart cells is maintained at a level higher than electrochemical equilibrium by a combination of NHE and SITS-

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III) Renal HCO3⫺ reabsorption. aNBC has a similar molecular physiology to mammalian NBCe1-A (338) and likely plays the same important role in HCO3⫺ reabsorption from the glomerular filtrate. However, the acidification of amphibian tubule fluid is predominantly achieved in the late distal tubule, by an electrogenic Na⫹-dependent process (749), as demonstrated by early experiments on Necturus and leopard frog (Rana pipiens) renal tubules (657) and later measurements of bicarbonate reabsorption in Ambystoma maculatum renal tubules (1088). FIGURE 14 shows models of the role of NBCe1 in bicarbonate reabsorption by amphibian pronephric epithelia in the proximal and distal tubules. Norepinephrine has an inhibitory effect upon NBCe1-mediated HCO3⫺ reabsorption in the Ambystoma PT, perhaps by elevating cAMP levels (2), a factor that is also inhibitory to electrogenic NCBT activity in rabbit renal tubule preparations (821).

MARK D. PARKER AND WALTER F. BORON sensitive NDCBE-like activities, both of which play a role in recovery from an acid load imposed by an NH4⫹ prepulse (594). In chicken chondrocytes, an NDCBE-like activity is expected to contribute to the recovery from intracellular acidosis that would accompany a mechanical load (218). Stilbene-sensitive NCBT activity has also been reported in chicken enterocytes (734) and colonocytes (146).

IV. GENERAL FEATURES OF NCBTs

A. General Structural Features of Mammalian NCBTs Because the Na⫹-independent Cl-HCO3 exchangers (i.e., AE1–3) in the Slc4 family are 28 –34% identical to NCBTs at the amino acid level (807), it is likely that NCBTs share many common structural features with AEs. Studies concerning the structure of NCBTs are therefore heavily supplemented by reference to the wealth of data produced by ongoing studies into the structure of AE1. However, because of their differing functions, crucial structural differences are likely to exist between the AEs and NCBTs (1112), and perhaps even among NCBTs. Here, using as our template a model of AE1 structure, refined with new data from recent studies of NBCe1 topology (see Refs. 1112, 1113 as well as FIGURE 2A), we consider the common structural features of mammalian NCBTs and note some key differences 1) between NCBTs and AE1 and 2) among individual NCBTs. Although this section is intended to refer specifically to mammalian NCBTs, many conclusions likely hold true for most vertebrate NCBT-like transporters, and even some invertebrate NCBTs, which are predicted to have a similar topology to mammalian NCBTs. The NCBTs are glycosylated membrane proteins with predicted nonglycosylated molecular weights of between 116 and 140 kDa. As shown in FIGURES 2A AND 15, each transporter has three major domains: a large 46 – 66 kDa cytosolic Nt, a ⬃60 kDa transmembrane domain (TMD) encompassing 12–14 TMs, and a smaller ⬃10 –14 kDa cytosolic Ct. FIGURE 15 shows the TMD with 13 ␣-helical spans plus an “extended structure” linking TM11 and TM13, as well as

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1. Oligomerization The detection of NBCe1 dimers in rat kidney sections (865) and NBCe1, as well as NBCe2, tetramers in the human embryonic kidney cell line HEK-293 (773) indicates that NCBTs, like AE1, form higher oligomers. Like AE1, NBCe1 molecules, and likely all NCBTs, form oligomers stabilized at multiple contact points. Oligomerization is presumed to be a prerequisite for functional expression of the transporter.24 In the case of AE1, homodimers are stabilized by Nt-Nt interactions (1091) as well as TMD-TMD interactions (791, 1023). AE1 tetramers are dimers of homodimers that are linked, at contact points in their Nt, by cytoskeletal proteins such as ankyrin (156). It is unknown whether NBCe1 tetramerization requires an accessory protein. Size-exclusion chromatography indicates that the isolated Nt of human NBCe1, human NBCe2, and rat NBCn1 all form homodimers (320), and preliminary X-ray diffraction data demonstrates that the NBCe1-Nt dimer is stabilized by interlocking arms (321), homologous to those that stabilize AE1-Nt dimers (1091). Evidence of TMD-TMD interactions (or perhaps even Nt-Ct interactions) within an NBCe1 dimer is provided by experiments in which an NBCe1 construct that lacks a Ct coimmunoprecipitates with an NBCe1 molecule that lacks an Nt (276). Unlike AE1 dimers, NBCe1 dimers are further stabilized by disulfide bridges between cysteine residues in the third extracellular loops (EL3 in FIGURE 15) of opposing monomers (471, 632, 834). Some evidence suggests that NBCe1 monomers within a dimer are capable of functioning independently (471). A concatameric NBCe1 molecule was created in which a mutant NBCe1 monomer [T442C, which can be selectively blocked with (2-sulfonatoethyl) methanethiosulfonate, also known as MTSES, a cysteine-reactive reagent] was joined to a wild-type monomer (WT, which is unaffected by MTSES). Unlike a WT-WT concatamer that is not inhibited by MTSES and a T442C-T442C concatamer that is 100% blocked by MTSES, hybrid concatamers are only 50% blocked, as if the WT monomer within the dimer operates independently of the blocked T442C monomer (471). Another possibility is that MTSES binding to the WT-T442C heterodimer produces a 50% blockade of each monomer within the dimer. 2. NCBT domain structure Based on the presence of alternating variable and conserved regions of protein sequence, we can consider each of the 24 We define functional expression as the product of surface expression and the intrinsic transporter activity of individual molecules.

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The human genome, and likely every mammalian genome, includes 10 SLC4 genes. Five of these have been unequivocally characterized as encoding NCBTs: Slc4a4 (NBCe1), Slc4a5 (NBCe2), Slc4a7 (NBCn1), Slc4a8 (NDCBE), and Slc4a10 (NBCn2) as displayed in FIGURE 3. Each has a distinct molecular action, distribution, and role, considered in section V. In the present section, we consider features that are common among NCBTs, including oligomeric state, domain structure (e.g., predicted topology, conserved sequence motifs), and maneuvers that inhibit or stimulate transport.

an extended, glycosylated extracellular loop (EL3) between TMs 5 and 6.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

7 EL3 C

C C

C

Lumen

1

Cytosol

5 Nt-TMD linker

2

Nt loop

5

6

7

8

9

10

11

E 13

14

Ct core 9

TMs6–14 8

Ct 4

Nt core 1

Ct appendage

10

HOOC

2 Nt appendage 1 NH2

FIGURE 15. Domains and subdomains of NCBTs. Representation of a typical NCBT showing the three domains (Nt, TMD, and Ct) together with the 10 numbered subdomains that comprise them. The common and unique features among NCBTs in each subdomain are discussed in the text. An annotated alignment of human NCBTs showing the division of subdomains is provided in Appendix I.

three major NCBT domains as being divided into a total of 10 subdomains (see diagram in FIGURE 15 and sequence alignments in Appendix I). The first five subdomains are all part of the Nt. The TMD includes the sixth (transmembrane spans 1–5, TMs1–5), seventh (third extracellular loop, EL3), and eighth (TMs 6 –14) subdomains. Finally, the Ct consists of two subdomains (a conserved core and a variable region). We now will discuss each domain and subdomain individually. Unless stated otherwise, the amino acid residue numbers, provided as a guide, refer to the human renal variant of NBCe1 (NBCe1-A; GenBank protein accession no. NP_003750; see guide to NBCe1 nomenclature below). A) THE CYTOSOLIC Nt. The Nt can be divided into five subdomains (Nt appendage, Nt core 1, Nt loop, Nt core 2, and Nt-TMD linker; FIGURE 15). The protein sequences of the appendage, loop, and linker subdomains of the Nt differ greatly among NCBTs, and often include splice cassettes (reviewed in Ref. 104). On the other hand, the protein sequences of the two core subdomains are well conserved among NCBTs and, according to the preliminary crystalstructure data (321) and by comparison to the crystal structure of the AE1 Nt (1091), form the structural core of the Nt. The core of the Nt exhibits considerable structural homology to certain bacterial EIIA proteins, a class that function as cytosolic regulators of membrane proteins.

In the case of the AEs, the Nt is not vital for either cellsurface presentation or transporter activity, but rather in-

cludes binding sites for protein partners and determinants that direct the trafficking of the transporter. Studies of NBCe1 suggest that the Nt of NCBTs is not vital for cell surface presentation of the rest of the molecule (276, 575, 634) but that it is required for NCBT activity (276, 634). Preliminary studies show that coexpression of an isolated NBCe1-A Nt enables the otherwise inactive NBCe1 TMD to perform electrogenic Na/HCO3 cotransport in Xenopus oocytes, indicating that the Nt is an activating binding partner of the TMD (724). Similarly the TMD of the human chloride channel ClC-1 is activated by its cytosolic domain (842, 1050). The mode of action by which the NBCe1 Nt activates the TMD is unknown. In the case of ClCs, the cytosolic domain is thought to act as a scaffold that influences the alignment of transmembrane spans within the TMD (283) as well as sensor that conveys information to the TMD (reviewed in Ref. 66). Structural studies of a ClC homolog from a red alga reveal an extensive interface between the transmembrane and cytosolic domains (283). I) Nt appendage (subdomain 1). The protein sequence of the Nt appendage is poorly conserved among NCBTs. Transcription from alternative promoters leads to great divergence in sequence and size of this subdomain (41–92 amino acids). The result may be protein variants with little or no homology in the affected region (e.g., NBCe1-A versus NBCe1-B) or variants with an effectively truncated Nt in which translation initiates at an otherwise “internal” Met residue (e.g., NDCBE-A versus NDCBE-C). Inasmuch

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3

4

TMs1–5 6

Nt core 2

Nt

3

TMD

MARK D. PARKER AND WALTER F. BORON as the electron density corresponding to sequence encoded by residues 1– 62 is sparse in X-ray diffraction data gathered from crystals of NBCe1-A Nt, it is likely that the Nt appendage is either loosely structured or is structured but tethered to the Nt core 1 subdomain by a flexible linker.

It is unknown how the ASD and AID exert their effects upon the NCBT TMD. The Nt appendage of NBCe2 is different from that of other NCBTs both in primary sequence and charge distribution, suggesting that NBCe2 may not contain a typical Nt AID or ASD. The Nt appendage also includes a number of potential phosphorylation sites, four of which–Ser89, Ser91, and Tyr92 of NBCn1-B (210, 385, 700) and Thr49 of NBCe1-B (345)– have been demonstrated to be phosphorylated in vivo. Thr49 is required for cAMP-induced activation of NBCe1-B, although phosphorylation of Thr49 is not (345). II) Nt core 1 (subdomain 2). This ⬃65-amino-acid-long region is intertwined in three dimensions with Nt core 2 and together the two subdomains form the core structure of the Nt. Over a quarter of the sequence in Nt core 1 consists of Glu/Asp residues. In AE2, the Nt residues that confer pH sensitivity to Cl⫺ transport are particularly concentrated in Nt core 1 (summarized in Ref. 907). Centrally positioned in Nt core 1 is the well-conserved “ETARWIKFEE” signature sequence, more precisely for NCBTs “W87[K/R]E[S/T]ARW[I/L]KFEE92”, that marks the start of conservation between NCBTs and AEs. By structural homology with the AE1 Nt, residues within this motif are predicted to form charge interactions with each other (Arg86 interacts with Glu92) and residues in Nt core 2 (Arg86 interacts with Lys227; Glu91 interacts with Arg298; see Refs. 166 and 577). Together, these residues are situated at one end of a “tunnel” of polar residues within the Nt that has been proposed to be part of an ion-permeation pathway (166). Speaking to the proposed importance of and interaction between Glu91 and Arg298 is the severe phenotype (discussed below) of the naturally occurring human mutation R298S (411). Mutation of ei-

838

One preliminary report suggests that a conserved Cys120 towards the end of Nt core 1 is important for the functional expression and oligomerization of NBCe1 (52). In all NCBTs, this cysteine falls within a region homologous to the first ␣-helix in the AE1 Nt, which in the three-dimensional structure is near but not at the Nt dimer interface (1091). In NBCe2 and AE2, this region contains a leucinezipper motif (542). III) Nt loop (subdomain 3). In the AE1 Nt, this variable region corresponds to a flexible “hinge,” including 10 residues that are not defined in the crystal structure, that is likely to be a loop that extends from the core structure of the Nt, linking the two core subdomains. In AE1, the Nt loop contains determinants of protein 4.2 binding (469) and ankyrin binding (169, 252). The Nt loop exhibits strong sequence conservation between NBCe1 and NBCe2 and among NBCn1, NBCn2, and NDCBE, but not between these two sets. In NCBTs, the Nt loop can vary in length due to the inclusion/exclusion of splice cassettes (e.g., cassette I of NBCe1, cassette II of NBCn1, and cassette A of NBCn2). As with the variable sequence in the Nt appendage, the splice cassettes within the Nt loop are nonessential to transporter function (201, 317), suggestive of a regulatory or protein-binding role for the Nt loop. Concordantly, a preliminary report suggests that cassette II of NBCn1 interacts with calcineurin A (715). IV) Nt core 2 (subdomain 4). This region, the second and longer conserved region in the Nt (encompassing ⬃132 amino acids), includes the interlocking arms that are critical for dimerization of the Nt (320, 1091). An AE1-Nt based homology model of the NBCe1 Nt predicts that Arg298 can form charge interactions with either of two residues that are adjacent in the three-dimensional structure, Glu91 in Nt core 1 and Glu295 in Nt core 2 (166). Finally, at least in the case of NBCe1, Lys227 is predicted to interact with residue Glu92 in Nt core 1 (166). As mentioned earlier, these polar residues line a tunnel within the Nt that is important for normal functioning of NBCe1. V) Nt-TMD linker (subdomain 5). A flexible linker joins the core structure of the Nt and the TMD. The majority of this region has a disordered structure in AE1 (1091) and is poorly conserved among Slc4s. In NBCe1 and NBCe2, this region contains an additional glycine-rich sequence that

25 Defects that reduce the intrinsic transporter activity of individual molecules.

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Reflecting its variable nature, most of the sequence within the Nt appendage is nonessential for NCBT activity (634, 718). However, this region can include such elements as: 1) the autostimulatory domain (ASD) of NBCe1-A, the inclusion of which stimulates NBCe1 activity; 2) the autoinhibitory domain (AID) of at least NBCe1-B and NBCn2, the inclusion of which inhibits NCBT activity; and 3) the IRBIT binding determinants (IBD) of at least NBCe1-B and NBCn2-B, and presumably also the IBD of NBCn1-B and NDCBE-B, the inclusion of which confers sensitivity of the NCBT to stimulation by the cytosolic protein IRBIT. Sequestration of the Nt AID may be one of the mechanisms by which IRBIT activates NCBTs (559, 718, 859).

ther Glu91 or Arg298 can cause trafficking and per-molecule transport defects25 in NBCe1 (166, 411, 577), whereas the complementary compound mutant E91R/R298E has near-normal activity (166).

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

B) THE TMD.

This region comprises three subdomains: transmembrane spans (TMs) 1–5, the large extracellular loop (EL3), and TMs6 –14 (FIGURE 15). Although neither TMs 1–5 nor TMs 6 –14 of AE1 or NBCe1 are capable of HCO3⫺ transport by themselves, when coexpressed as two separate fragments in Xenopus oocytes, TMs 1–5 and TMs 6 –14 are capable of self-associating to recreate the transport activity of the full-length protein (AE1 or NBCe1, see Refs. 353 and 723). The TMDs of NCBTs have a high degree of sequence identity. A high-resolution crystal structure has yet to be reported for the transmembrane domain of any Slc4 family member, but topology models predict a 10 –14 TMs together with the hydrophilic loops that link them (48, 302, 1116). Although one group had proposed 10-TM model (950), new preliminary data generated by probing the chemical accessibility of introduced cysteine residues (1114, 1115) are consistent, between TM1–TM8, with the model in FIGURE 2. For the results of extensive mutagenesis studies that highlight residues within this domain important to NBCe1 folding and function, we refer the reader to studies from the Kurtz laboratory (e.g., Refs. 5 and 1112). As noted above, the TMD of NBCe1, plus its Ct, is capable of trafficking to the cell membrane without the Nt, yet it is nonfunctional (276, 634). The TMD also includes determinants for the electrogenicity/electroneutrality of transport cycles (178, 179, 193). I) TMs 1–5 (subdomain 6). Sequence conservation between NCBTs and AE1 extends throughout the first five putative TM spans, which are linked by short, hydrophilic loops. TM1 contains residues that appear to lie in the ion-translocation pathway. Indeed, in a study employing cysteinescanning mutagenesis, residues predicted to map along one edge of a TM1 helix were targets of Cys-reactive agents that blocked transport activity (1110).

By homology to AE1, TM2 and TM3 may form a re-entrant loop that is stabilized in the membrane more by interactions with surrounding TMs than by protein-lipid interactions (188). Thus TM2 and TM3 may not be topogenic without TM1 and TM4 in place (188, 703). The observation that two neighboring mutations in TM3–G485S and G486R, both associated with proximal renal tubular acidosis (pRTA)– cause per-molecule defects in NBCe1 without apparently affecting protein delivery to the plasma membrane (393, 576, 929, 930) suggests that TM3 residues are important for ion translocation. The extracellular end of TM5 leading into the third extracellular loop contains a conserved lysine (807), Lys559 in NBCe1. In NBCe1, this Lys residue is the second K in the motif “KKMIK,” which plays a critical role in both the reversible and irreversible interaction of disulfonic stilbenes that inhibit anion transport (611). The determinants of stilbene inhibition are discussed in greater detail below; other compounds known to inhibit NCBT activity are considered. Although all five NCBTs retain this Lys, the Na/HCO3 cotransport activity of NBCn1 is relatively insensitive to blockade by DIDS. II) EL3 (subdomain 7). The third extracellular loop of NCBTs is an extended region (⬃86 amino acids) that links TMs 5 and 6. The integrity of this loop is not vital for NCBT activity (Boron lab, unpublished data; see Ref. 723). Moreover, a 9-amino acid hemagglutinin epitope-tag can be introduced into EL3 of NBCe1 without disruption of NBCe1 activity (634). Despite a high degree of sequence conservation between NBCe1 and NBCe2 and among NBCn1, NBCn2, and NDCBE in this region, the only globally conserved motifs are a series of consensus N-linked glycosylation sites and four cysteine residues. We will consider these two features in the following paragraphs. NBCe1 (190, 515), NBCn1 (174), NBCn2 (177, 755), and NDCBE (176) are all glycosylated in vivo, as evidenced by an increase in gel mobility upon PNGase F treatment. Human NBCe1 and NBCn2 have three sites, human NBCn1 and NBCe2 have four, and human NDCBE has only two. All are within EL3; however, not all of the N-glycosylation sites in an NCBT may actually be glycosylated. In the case of NBCe1, which has three putative sites, only the distal two sites of the form “Asn-Xaa-Thr,” but not the proximal “Asn-Xaa-Ser” site, are normally glycosylated in Xenopus oocytes (190) (Xaa is a 3-letter placeholder for any amino acid). Glycosylation does not appear to be vital for the NCBT function of NBCe1 (190), but a mutant NBCn2 in which all three glycosylatable Asn residues are replaced by Gln, exhibits poor protein expression compared with the wild-type transporter (177). Within an NBCe1 dimer, the four conserved cysteine residues (Cys583, Cys585, Cys630, and Cys642) form disulfide

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may confer some extra flexibility between the Nt and TMD, although a precise role for this region has not yet been described. The length of the Gly-rich region in NBCe2 (23 Gly in a stretch of 30 residues in human NBCe2) varies among mammalian species, is reduced to three or four Gly residues in zebrafish NBCe2 isoforms, and is absent altogether in the predicted protein sequence of Xenopus tropicalis NBCe2. Conservation among NCBTs returns close to the start of TM1 at the D405IKRK409 motif, which is homologous to the protein-4.1–interaction motif in AE1 (452). Indeed, 4.1B and NBCe1 are colocalized in, and can be coimmunoprecipitated from, murine PT epithelia (957, 958). The NBCe1 protein complex also includes the membrane-associated guanylate kinase homolog p55 (957), which can act as cytoskeletal anchor (reviewed in Ref. 41). Asp405 as well as Asp416 in the conserved portion of the linker are critical for plasma membrane targeting of NBCe1 (574).

MARK D. PARKER AND WALTER F. BORON bonds. Cys583 and Cys585 form an intramolecular disulfide bond with each other, and Cys630 and Cys642 intermolecularly bond to their counterparts within an NBCe1 dimer (1111).

C) THE CYTOSOLIC Ct. The Ct (90 –105 amino acids) consists of

two subdomains: a conserved region (Ct core) and a variable region (Ct appendage). The variable region is the site of extensive variation in splicing, which potentially enables each transporter to interact with a variety of protein binding partners. As evidenced by studies on NBCe1-A and NBCn1, determinants within the Ct are vital for the stable plasma membrane expression of NCBTs (e.g., Refs. 276, 578, 603, and 930). A study of the isolated Ct domain of NBCn1 (603) indicates that this domain is relatively unstructured, although a subsequent study of a smaller peptide corresponding to sequence within the NBCe1 Ct reveals some ␣-helical content (573). I) Ct core (subdomain 9). The protein sequence of the first subdomain of the Ct is well conserved among NCBTs and includes three notable motifs that are discussed below: 1) a dihydrophobic trafficking signal, 2) aspartate clusters, and 3) lysine clusters. II) FL targeting motif. An “FL” sequence in the Ct of NBCe1 is necessary for the basolateral presentation of the transporter, and is located in a region determined by CD spectroscopy to have some ␣-helical content (573). Deletion of the last 92 amino acids (thereby deleting the “FL” motif) of NBCe1-A causes the protein to be destabilized in the

840

III) Asp clusters. A motif similar to the “LDADD” sequence in AE1 has been reported to be important for CA II binding and consequently NBCe1 activation (68, 350, 604, 764), the CAII metabolon hypothesis. Furthermore, expression of NBCn1 is reported to cause a redistribution of CAII to the plasma membrane of HEK cells (604). However, in subsequent studies by others, peptides corresponding to human AE1, NBCe1, or NDCBE Ct do not bind CA II in vitro (748) and a CA II-dependent activation of NBCe1 cannot be demonstrated by co-expressing NBCe1 and CA II in Xenopus oocytes (1063), by coinjecting purified CA II into NBCe1-expressing oocytes (613), or by expression of an NBCe1-CA II fusion protein (613). The evidence presented in favor and against a physiologically relevant interaction between NCBTs and CA II was recently evaluated in Reference 102. IV) Lys clusters. Following the Asp cluster is a long stretch of charged residues that are characteristic of the NCBTs but not the AEs. The most striking example is in NBCe1, which has a string of 17 consecutive charged residues, 12 of which are lysines. V) Ct appendage (subdomain 10). The terminal subdomain of the Ct can vary greatly among NCBT isoforms. Some of the features that can be included or excluded by alternative splicing in this region are listed below. A) Arg-based ER localization signals. The Ct of both NBCe1-A and NBCe2c contain “R-X-R” sequences that, in many other transporters, prevents forward trafficking of transporter molecules until the signal is masked by oligomer formation, or interaction with a binding partner (recently reviewed in Ref. 644). The relevance of this motif in NCBTs has yet to be tested, but it is notable that a truncated NBCe1-A that lacks the last 65 amino acids of the Ct (including an “RER” sequence) has a dominant-negative effect on the forward trafficking of full-length NBCe1-A molecules (930). B) Motifs for binding PDZ domains. A class I PDZ-domain– binding sequence, conforming to an “-ET[T/S/C]L” consensus (874, 992), is common to NBCe1-C (79), NBCn1 (769), and NBCn2-C/D (317) and mediates interactions between the transporter and cytoskeletal scaffolding proteins such as NHERF1 (562, 711, 769), harmonin (790), and PSD-95 (780). These binding partners serve as foci for clustering of membrane proteins, such that NCBTs may associate with the vacuolar-type H⫹-pump (769), the N-

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III) TMs 6 –14 (subdomain 8). Global conservation of protein sequence among Slc4s continues throughout the remainder of the TMD. Gly723 in the fourth extracellular loop (EL4) of NBCe1 is reported to be necessary for interaction with CA IV (32). This loop also contains determinants of transporter action, inasmuch as the electrogenicity versus electroneutrality of chimeric NBCe1/NBCn1 transporters depends on the origin (NBCe1 versus NBCn1) of EL4 (178). A cysteine residue (Cys916) at the putative intracellular end of TM12 is palmitoylated in the related transporter AE1 (698). However, this Cys residue is not necessary for the function or surface expression in heterologous systems of either AE1 (154, 465) or NBCe1 (471). Cysteine-scanning mutagenesis studies suggest structural differences between AE1 and NBCe1 in this region (1112) and that residues in TM8 of NBCe1 lie along the ion-translocation pathway (633). Of particular interest in this region are Leu750 in TM8 that appears to lie in a conformationally sensitive part of the transporter (633) and the pRTAassociated residue Ala799 in the vicinity of TM9/TM10, mutation of which causes per-molecule transport defects and elicits an unusual DIDS-stimulated, HCO3⫺-independent conductance in NBCe1 (721) that is similar to that observed for NBCn1.

basolateral membrane and to be mistargeted to the apical membrane of an opossum kidney cell line (276). A study in Xenopus oocytes demonstrates that deletion of the last 41 amino acids (deleting the “FL” motif) from NBCe1-A is sufficient to cause near-total intracellular retention of the transporter (634).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

methyl-D-aspartic acid (NMDA) receptor (780), and pertinent to the infrequent examples of apical NCBT localization, CFTR (711). The extreme Ct sequence of NBCe2c “-SYSL” has been suggested to resemble a class II PDZdomain ligand (768), conforming to a “-X-␸-X-␸” consensus (874). No currently identified NDCBE variant terminates with a consensus PDZ-binding domain. A truncated NBCn1 that lacks only the PDZ-domain binding sequence traffics to the plasma membrane of HEK cells and mediates a similar transport activity to full-length NBCn1 (711). Thus, at least in heterologous systems, the PDZ-binding motif is not critical for functional expression of NCBTs.

B. Inhibition and Stimulation of NCBTs 1. NCBT inhibition NCBTs are amenable to blockade although chemical inhibitors specific to any one native NCBT has yet to be reported. Thus the pharmacological tools for distinguishing NCBTs from each other are currently lacking. On the other side of the coin, an NCBT activity that is not stilbene sensitive typically correlates with the presence of a single NCBT, namely NBCn1. Furthermore, blockade of selected NCBT molecules can be achieved by mutagenic introduction of inhibitor binding sites (e.g., Ref. 471) and the use of specific antibodies or antisense probes appear to be promising methods for effective knockdown of specific NCBTs (see below). Demonstrated NCBT inhibitors and methods of NCBT inhibition are listed below and the chemical structures of pharmacological agents mentioned are presented in TABLE 4. Interventions that downregulate NCBT transcription, translation, and activity in vivo are discussed for each NCBT in section V. A) STILBENE DISULFONATES.

All NCBTs, with the exception of NBCn1, are inhibited by stilbene derivatives such as DIDS

As is the case for AE1 (144, 569), inhibition of NBCe1 by DIDS is temporally biphasic (611). The first phase is a rapid, reversible component of inhibition that presumably reflects an ionic interaction with the protein. In the case of NBCe1-A, this inhibition depends to a large extent on three lysine residues, in the motif KKMIK—located at the putative extracellular end of TM5. Replacing all three Lys residues with either Asn or Asp results in a 10-fold or more increase in Ki, and replacing with three Glu residues results in a 20-fold increase (611). However, it is the second lysine (K559 for human NBCe1-A) that is the most important of the three for DIDS inhibition and, indeed, only K559 that is conserved among all five human NCBTs. However, the influence of this lysine must be context dependent, inasmuch as the poorly DIDS-sensitive NBCn1 includes the TM5 motif “EKLFD”.28 A preliminary report suggests that mutating this motif to “EKLFK” renders the Na/HCO3 cotransport activity of NBCn1 readily inhibitable by 500 ␮M DIDS (191). The second, slower phase of inhibition by extracellular DIDS is irreversible and presumably reflects the covalent reaction of the bifunctional DIDS molecule with the –NH2 moiety of one or more Lys residues, although in principle the electrophilic isothiocyanate groups of DIDS could derivatize the nucleophilic side chains of other amino acid residues, such as Cys, His, Ser, or Tyr (622). Because irreversible DIDS inhibition still occurs with the mutants NNMIN and RRMIR, the covalent reaction requires additional determinants elsewhere in the molecule that have yet to be identified (611). DIDS is also capable of inhibiting NBCe1 when applied to the intracellular side of the protein in cell-detached plasma membrane patches (264, 381, 634).

26 Substituted stilbenes undergo cis-trans photoisomerization, the cis isoforms being less potent than the trans isoforms as Slc4 inhibitors (848, 880, 1001). 27 In fact, the Na⫹ conductance mediated by NBCn1 is stimulated after prolonged exposure to 500 ␮M DIDS (189, 201). 28 This motif is “KLFH” in mouse and rat orthologs of NBCn1.

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C) Autoinhibitory sequence. Alternative splicing of NBCe1 at its extreme Ct can result in the inclusion of a 46-amino-acid appendage (in NBCe1-A/B) or a 61-amino-acid appendage (in NBCe1-C). Although NBCe1-B and NBCe1-C have similar intrinsic activities (634), NBCe1-C has a greater activity than NBCe1-B when the Nt auto inhibitory domain of both is neutralized by Nt truncation (634) or by IRBIT coexpression (967). It is unknown to what extent the 61-amino acid appendage exerts a stimulatory effect or the 43-amino acid appendage exerts an inhibitory effect. Similarly, alternative splicing of NDCBE at its extreme Ct can result in the inclusion of a 17-amino acid Ct sequence (see NDCBE-B/D below) that is inhibitory to the functional expression of the transporter (717).

(337, 835, 1009, 1021) and DNDS (634).26 DIDS is a disulfonic stilbene (i.e., it has 2 negative charges). DIDS blocks the HCO3⫺-dependent conductance mediated by NBCe1 (611) in Xenopus oocytes with an apparent Ki of ⬃40 ␮M (596, 611), which is less potent than its blockade of AE1 in oocytes (Ki ⬃6 ␮M; Ref. 1044). In an exploratory setting, we consider 200 ␮M DIDS to be an appropriate experimental concentration to achieve a substantial block of the activities of NBCe1, NBCe2, NDCBE, and NBCn2. NBCn1 is unique inasmuch as it is poorly inhibited by even 500␮M DIDS.27

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596, 611

264, 611

596

Ki ⫽ 36-40 ␮M when applied to oocytes expressing NBCe1. Also blocks NBCe2, NDCBE, and NBCn2 (typically used at 200 ␮M). Blockade of NBCn1 is poor even at 500 ␮M.

Ki ⫽ 13-25 ␮M when applied to oocytes expressing NBCe1. Also blocks NBCe2. Untested on electroneutral NCBTs.

Ki ⫽ 100 ␮M when applied to oocytes expressing NBCe1. Untested on other NCBTs.

Stilbene disulfonate (e.g., DIDS; trans isomer depicted)

Tenidap

Niflumic Acid

Continued

Reference Nos.

Chemical Structure

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842 Application

Name

Table 4. NCBT inhibitors

MARK D. PARKER AND WALTER F. BORON

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Detailed information concerning the use of the NCBT inhibitors listed in this table, together with a list of drugs that have been suggested but not demonstrated to act on NCBTs, is provided in the text. Chemical structures were drawn using ChemBioDraw Ultra version 12.0 (Perkin Elmer, Akron, OH).

247

50 ␮M is sufficient to block NCBT-like activity in cholangiocarcinoma cells. Untested on heterologously expressed NCBTs.

S3705

Not disclosed

160, 545

Ki ⫽ 1.7 ␮M when applied to ventricular myocytes, which express at least NBCe1 and NBCn1. 10-30 ␮M ⫺ blocks Na⫹-dependent HCO3 transporters in tumor cells which express at least NBCn1. Untested on heterologously expressed NCBTs.

S0859

596

Reference Nos.

264

Ki ⫽ 10 ␮M when applied to oocytes expressing NBCe1. Untested on other NCBTs.

Application

500 ␮M effects a total block of NBCe1 currents when applied to the cytosolic face of oocyte membrane patches. Untested on other NCBTs and on the extracellular face of NBCe1.

Chemical Structure

Benzamil

diBAC oxono(e.g., diBA(3)C4)

Name

Table 4.—Continued

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

843

MARK D. PARKER AND WALTER F. BORON Tenidap29 blocks, but with only partial reversibility, at least NBCe1 (Ki ⬃15–25 ␮M; Refs. 264, 611) and NBCe2 (869). Niflumic acid, an NSAID often used to inhibit anion channels, blocks at least NBCe1 (Ki ⬃100 ␮M; Ref. 596). B) NONSTEROIDAL ANTI-INFLAMMATORY DRUGS.

porter antibody (74). The antibody reacted on western blots with a 56-kDa protein and apparently blocked NBCe1 activity (74). However, after the cloning of NBCe1, we now appreciate that the immunoreactive protein is too small to have been NBCe1-A. Thus the blockade must have been indirect. The identity of the 56-kDa protein is unknown.

C) DIBAC OXONOL DYES. These fluorescent, voltage-sensitive dyes block at least NBCe1 (Ki ⬃10 ␮M; Ref. 596).

H) ANTISENSE PROBES.

D) BENZAMIL. This analog of amiloride effects a complete, yet reversible block of rat NBCe1 when applied to the cytosolic surface of oocyte membrane patches at 500 ␮M (264).

I) PHOSPHATASES.

F) S3705. This agent, when applied at a concentration of 40 ␮M,

blocks at least the NCBT activity present in breast carcinoma (1041) and cholangiocarcinoma (247) cell lines, slowing tumor growth and, in the latter case, promoting apoptosis. Although both studies report blockade of NDCBE-like activity, the molecular identities of the NCBT responsible are not demonstrated. These cell lines likely express at least NBCn1 (546) as well as a stilbene-sensitive NCBT. G) ANTIBODIES.

An alternative approach to target specific NCBTs involves the use of inhibitory antibodies directed against extracellular epitopes, or to reduce NCBT transcript, and consequently protein, abundance via antisense technology. In two studies, the action of NBCe1 was inhibited using antibodies raised against an epitope in the third (i.e., longest) extracellular loop of NBCe1 (225, 481). In a third study, conducted prior to the cloning of NBCe1, rabbit proximal tubule vesicles enriched for Na/HCO3 cotransporter activity were used to raise an anti-Na/HCO3 cotrans29

Developed by Pfizer Inc (New York, NY). Developed by Sanofi-Aventis U.S. LLC (Bridgewater, NJ). A synthesis protocol based on commercially available compounds has been developed by Larsen and co-workers and is provided in Ref. 545. 31 Bachmann et al. cite unpublished observations from Aventis Laboratories that S0859 blocks NBCe1 with a Ki ⬃6 ␮M, but does not block NBC2/3 (i.e., NDCBE) as expressed in CHO cells. 32 The use of S0859 as an “NBC1 blocker” (849) or an “NBCn1 inhibitor” (546) in cells in which these transporters are the dominant NCBT paralog does not constitute a demonstration of paralog specificity of the compound. 30

844

The time-dependent rundown of NBCe1-A activity in excised Xenopus oocyte macropatches can be slowed by maneuvers that inhibit protein phosphatase activity, indicating that at least NBCe1-A can be inhibited by dephosphorylation (1049).

J) INTRACELLULAR MAGNESIUM.

In bovine parotic acinar cells, as well as in mammalian cell lines overexpressing NBCe1-B, NBCe1 activity is inhibited by intracellular Mg2⫹ (1066). Although this effect has not been demonstrated to be direct, a mutant NBCe1 construct that lacks the Nt sequence specific to NBCe1-B (a region that includes the AID as well as IRBIT binding determinants) exhibits a substantially reduced sensitivity to Mg2⫹ (1066). In HEK cells, the coexpression of IRBIT also reduces the Mg2⫹ sensitivity of NBCe1-B (1067). K) MOLECULAR BIOLOGICAL APPROACHES. The per-molecule activity of NBCe1 is reduced by the removal of the Nt ASD, or the inclusion of the Nt AID (634). The per-molecule activity of NDCBE is reduced by the inclusion of the Ct AID (717). L) AGENTS SUGGESTED, BUT NOT PROVEN, TO BLOCK NCBTs.

The anticonvulsant levetiracetam (aka Keppra) and the diuretic hydrochlorothiazide (HCTZ) have both been reported to inhibit NCBT activities in isolated tissues (567, 571), but evidence of direct interaction of these drugs with NCBTs is presently lacking. Although direct blockade of NCBTs would indeed be anticonvulsive, the properties of levetiracetam instead appear to be a consequence of its interaction with synaptic vesicle protein 2 (SV2A; see Ref. 618). The possibility that levetiracetam exerts an indirect effect on the functional expression of NCBTs cannot be excluded. The psychoactive alkaloid harmaline blocks Na⫹-coupled transporters, perhaps by interaction with Na⫹-binding determinants (47, 864). When applied at 200 ␮M, harmaline is reported to effect a near-total, yet reversible block of at least 1) the Na⫹ and HCO3⫺ dependent pHi recovery in HEK cells expressing human NBCe1 (39), 2) the electrogenic NCBT activity in salamander Müller cells (680), and 3) the HCO3⫺-dependent Na⫹ flux in basolateral membrane vesicle preparations from rat (332) and rabbit (893, 897) renal cortex. However, 200 ␮M harmaline does not substantially inhibit human NBCe1-A expressed in Xenopus

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This drug30 blocks at least the NCBT activity present in 1) colonic crypt cells (likely a combination of NBCe1 and NBCn1 action, see Ref. 55),31 2) ventricular myocytes (likely a combination of NBCe1, NBCe2, and NBCn1 action, see Ref. 160), 3) coronary endothelial cells (likely a combination of NBCe1 and NBCn1 action, see Ref. 523), and 4) mammalian tumor cell lines (likely NBCn1, see Ref. 545). Thus S0859 may be the only reported potent inhibitor of NBCn1. S0859 may be more specific than stilbene derivatives, inasmuch as it is reported to be ineffective at blocking Na⫹-independent Cl-HCO3 exchanger activity (160). There are presently no reports of S0859 action on any of the five NCBTs expressed in isolation.32 E) S0859.

shRNAs, siRNAs, and hammerhead ribozymes have been used to reduce the abundance of specific NCBTs (90, 546, 593, 642, 989).

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

oocytes (Lee, Boron, and Parker, unpublished data), and thus blockade of NCBT activity by harmaline in renal membranes may be an indirect effect of blockade of other Na⫹dependent transporters. 2. NCBT stimulation Physiological stimuli that enhance transcription, translation, and activity of individual NCBTs in vivo are discussed, for each NCBT, in section V. Other maneuvers that enhance the activity of NCBTs are listed below.

Injection of PIP2 doubles the functional expression of NBCe1-B and NBCe1-C in intact oocytes, via a pathway that can be mimicked by IP3 injection and/or elevation of cytoplasmic [Ca2⫹] (968). The stimulation of NBCe1-B/C by IP3 injection is blocked by the kinase inhibitor staurosporine, consistent with the involvement of endogenous kinase activity (968). B) G PROTEIN–COUPLED RECEPTOR AGONISTS. Exposing oocytes to lysophosphatidic acid (LPA), which binds to endogenous LPA receptors and presumably acts via a pathway mimicked by PIP2/IP3 injection (462), increases the per-molecule activity of exogenously expressed NBCe1-C (968). A nuance is that LPA application, but not PIP2/IP3 injection, also increases the plasma membrane abundance of NBCe1-B (but not NBCe1-C) via a Ca2⫹-independent mechanism (968). C) ANTI-NBCe1 ANTIBODIES.

Application of an antibody raised against EL4 of NBCe1 stimulates NBCe1-like activity in myocytes (225).

D) INCLUSION OF AUTOSTIMULATORY SEQUENCE.

Full-length NBCe1-A exhibits a twofold greater activity than an NBCe1 construct that lacks the Nt ASD (559, 634). It is possible, although untested, that all NCBTs would also be stimulated by the replacement of their Nt appendages with NBCe1 autostimulatory sequence. Note that autostimulatory sequences specific to NBCe2, NBCn1, NDCBE, or NBCn2 have yet to be reported.

E) REMOVAL OF AUTOINHIBITORY SEQUENCE.

Truncation of the Nt AID from NBCe1-B/C and NBCn2 (634, 718) or truncation of the Ct AID from NDCBE-B/D (717) increases NCBT activity.

F) IRBIT. This soluble, 60-kDa protein is an important activator of certain NCBTs. Coexpression of IRBIT with NBCe1-B, NBCn1-B, NBCn2-B, or NDCBE-B (722, 881,

In mammalian cells, the effect of IRBIT upon NBCe1-B is twofold. In addition to stimulating the per-molecule activity of NBCe1-B, IRBIT, by antagonizing the WNK/SPAK signaling pathway, also causes an increase in plasma membrane abundance of NBCe1-B (1075). Other IRBIT binding partners include CFTR (1076), the IP3 receptor (43), NHE3 (371), and the cleavage and polyadenylation specificity factor CPSF (483).

V. NCBTs IN MAMMALS Each of the five mammalian NCBTs–Slc4a4 (NBCe1), Slc4a5 (NBCe2), Slc4a7 (NBCn1), Slc4a8 (NDCBE), and Slc4a10 (NBCn2)–plays a vital and unique role in acid-base homeostasis, and each has been the subject of much investigation. The two major roles played by NCBTs, reviewed here in section V, are 1) maintenance of pHi, local extracellular pH, interstitial pH, and plasma pH within a normal range (all NCBTs); and 2) support of transepithelial anion and fluid movement (e.g., NBCe1 and NBCn1 in salivary gland epithelia). We will detail crucial similarities and differences in the action, distribution, and role of each transporter, with the caveat that not all information on a particular NCBT may be transferrable among all mammalian species. For example, in one recent comparative analysis, NBCe1 transcripts were noticeably more abundant in preparations from human duodenum, than in equivalent samples isolated from mice and rats (485). In this section, we consider first the two electrogenic NCBTs (NBCe1 and NBCe2), and then the three electroneutral NCBTs (NBCn1, NDCBE, and NBCn2), these groupings reflecting the relatedness of the two major groups of NCBTs in FIGURE 3 AND TABLE 2. In section VI, we discuss the AEs (encoded by Slc4a1–3) and two other related Slc4-family members, Slc4a9 and Slc4a11. Also noteworthy, although not discussed further in this review, is the presence, in rat medullary thick ascending limb (mTAL) cells, of a stilbenesensitive, electroneutral K/HCO3 cotransport mechanism

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A) PIP2. Application of PIP2 to the intracellular face of excised Xenopus oocytes patches containing NBCe1-A stimulates transport (1049). However, PIP2 is rapidly hydrolyzed when injected into intact oocytes, an observation that may explain why injection of PIP2 does not result in the stimulation of NBCe1-A in whole cells (968).

1067), nNCBTs with defined autoinhibitory domains in the Nt, stimulates NCBT activity, in part by binding to the Nt and relieving transporter autoinhibition (559, 881). IRBIT must undergo a series of phosphorylations to become active, although the optimal phosphorylation state remains undefined (246, 881). Maximal stimulation of NBCe1-B activity by IRBIT, greater than that achieved by removal of the Nt AID, can be accomplished using a potent mutant IRBIT that lacks a PP-1 docking site and thus presumably becomes suitably phosphorylated (559). IRBIT has no effect on NBCe1-A because this variant has an autostimulatory domain but neither an IRBIT binding site or autoinhibitory domain (559, 634, 881).

MARK D. PARKER AND WALTER F. BORON (570), which has yet to be attributed to the activity of a specific transporter.

A. Mammalian Electrogenic NCBTs: NBCe1 and NBCe2 Of the five mammalian NCBTs, only NBCe1 and NBCe2 perform electrogenic Na⫹/HCO3⫺ cotransport. The molecular actions of NBCe1 and NBCe2 appear to be virtually indistinguishable; the major differences between NBCe1 and NBCe2 reside in their distribution and perhaps in their means of regulation. 1. NBCe1 (Slc4a4)

In keeping with these roles, NBCe1 dysfunction is associated with alterations in neuronal excitability (e.g., epilepsy), fluid-movement defects (e.g., corneal edema), and acid-base disturbances (e.g., proximal renal tubular acidosis or pRTA). NBCe1 has five known variants (-A through -E). NBCe1-A is the constitutively active renal variant. NBCe1-B, -C, and probably also -E are stimulated by the soluble protein IRBIT. NBCe1 is upregulated in acidosis and hypercapnia, conditions in which the action of NBCe1 would raise [HCO3⫺] in the blood plasma and/or intracellular fluid, thereby stabilizing pH. However, in some instances, the obligatory influx of Na⫹ that is coupled to the movement of HCO3⫺ into cells can contribute towards ischemic damage. NBCe1 is downregulated in conditions such as alkalosis and Na⫹ loading, when the requirement for NBCe1 action is reduced. B) NOMENCLATURE OF Slc4a4 PRODUCTS.

The nomenclature of Slc4a4 products has gradually evolved over the last 15 years. The original Slc4a4 gene-product, cloned from salamander kidney, was termed simply NBC for Na bicarbonate cotransporter (809). Prefixes have been variously added to this acronym to reflect either the genus of animal from which the NBC was cloned (e.g., rNBC was used to refer to rat Slc4a4 products), the organ from which the NBC was

846

Despite the multiplicity of acronyms, only five mammalian NBCe1 variants have been described to date. The current nomenclature defines Slc4a4 products as follows: NBCe1-A (previously known as rNBC, aNBC, kNBC, kNBC1, hkNBCe1), NBCe1-B (previously known as pNBC, pNBC1, rpNBC, hhNBC, hcNBC, rb1NBC), NBCe1-C (previously known as bNBC1, rb2NBC), NBCe1-D, or NBCe1-E. The unique and common features of each of these variants are discussed below. C) MOLECULAR ACTION OF NBCe.

I) Physiological substrates. NBCe1 was the first NCBT to be cloned from mammals (138, 140, 806, 808) and, like its amphibian ortholog, catalyzes the cotransport of 1 Na⫹ with 2 or 3 HCO3⫺ equivalents (FIGURE 16), resulting in the net movement of negative charge in the direction of net transport. There are a number of potential mechanisms by which electrogenic Na/HCO3 cotransport could occur. Preliminary reports suggests that it is CO32⫺, rather than HCO3⫺, that is the transported anion when the transporter is working with a 1:2 stoichiometry (336, 560), as shown in FIGURE 16B. It cannot be ruled out that the NaCO3⫺ ion pair is the transported anion (FIGURE 16C as well as Refs. 16 and 448), although potential evidence against this possibility are as follows: 1) the poor Li/HCO3 cotransport activity of NBCe1 that indicates a stronger cation selectivity than might be achieved in the case of NaCO3– versus LiCO3⫺ (39) and 2) the reported inhibition of NBCe1 by harmaline and inhibition by benzamil, drugs that interact with Na⫹ binding sites. Kinetic studies in other animals indicate that squid NDCBE, but not frog NBCe1, can transport the NaCO3⫺ ion pair. II) Apparent stoichiometry shift. NBCe1 certainly can function with a Na⫹:HCO3⫺ stoichiometry of 1:2 and appears capable of operating with a stoichiometry of 1:3. Stoichiometry plays a pivotal role in determining the direction of net transport. With an NBCe1 stoichiometry of 1:2, and

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A) SUMMARY. The electrogenic Na/HCO3 cotransporter NBCe1 (encoded by the Slc4a4 gene) is present in many organ systems throughout the body but is notably abundant in the following: 1) plasma membranes of neurons and glia in the central nervous system, where changes in pHi and pHo modulate neuronal excitability; 2) basolateral membranes of secretory epithelia, where NBCe1 mediates a HCO3⫺ influx that supports luminal HCO3⫺ secretion; and 3) basolateral membranes of renal PT cells, where NBCe1 mediates a HCO3⫺ efflux that is critical for the secretion of H⫹ into the tubule lumen, one consequence of which is HCO3⫺ reabsorption. This activity helps maintain a normal plasma [HCO3⫺].

cloned (e.g., kNBC was used to refer to kidney Slc4a4 products), or both (e.g., rkNBC was used to refer to rat kidney Slc4a4 products). In a case where more than one Slc4a4 splice variant was identified in an organ from a particular species, some authors added a number to the prefix (e.g., rb1NBC and rb2NBC were used to refer to two distinct products of the Slc4a4 gene from rat brain). Following the cloning of a cDNA from a second NBC-encoding gene, the original NBC was referred to as NBC1 (e.g., kNBC1 and pNBC1 distinguished kidney and pancreas Slc4a4 products) and the new ones given higher numbers (and not always different ones). Finally, with the cloning of electroneutral NBCs, a lowercase “e” for electrogenic was inserted into the acronym (e.g., NBCe1 and NBCn1 distinguish the electrogenic Slc4a4 gene-product from the electroneutral Slc4a7 gene-product) and thus the original, electrogenic Na/HCO3 cotransporter was finally renamed NBCe1 (100).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

NBCe1 or NBCe2 in 1:2 mode Na+

HCO3– HCO3–

A

Na+

CO3–

B

NaCO3–

C

Na+

HCO3–

D

H+ NBCe1 or NBCe2 in 1:3 mode H+

E

HCO3– HCO3– HCO3–

Na+

G

CO32–

NaCO3–

H

HCO3–

HCO3–

Na+

I

HCO3– HCO3–

Na+

H+

J

CO32–

NaCO3–

FIGURE 16. Molecular action of electrogenic NCBTs. Possible molecular mechanisms by which an electro⫺ genic NCBT could operate with an apparent Na⫹:HCO3 stoichiometry of 1:2 (A–D in top panel) and 1:3 (E–J in 2⫺ ⫺ bottom panel). Note that we have not considered any models that are based on CO3 -HCO3 exchange ⫺ (considered for NBCn1 in FIGURE 30), or models of HCO3 -stimulated electrogenic Na-2H exchange. We have also omitted mechanisms of 1:3 stoichiometry that could result from modification of model D.

typical ion concentration and voltage profiles across the membrane, Vm is more positive than the reversal potential and thus NBCe1 mediates a net influx of HCO3⫺ equivalents (for thermodynamic calculations, see examples in Refs. 103, 339, and 349). With a stoichiometry of 1:3, however, Vm would be more negative than Erev so that NBCe1 would mediate a net efflux of HCO3⫺. In astrocytes (75), parotid acinar cells (1065), corneal endothelial vesicles (543), pancreatic duct cells (344), ventricular myocytes (14, 1006) or when overexpressed in Xenopus oocytes (381, 853) and HEK cells (870), NBCe1 operates with a 1:2 stoichiometry, mediating net Na⫹ and HCO3⫺ influx (FIGURE 16, A–D, most likely FIGURE 16B). One study reports that NBCe1 also operates with a 1:2 stoichiometry in rabbit PTs (858) (where NBCe1 mediates net Na⫹ and HCO3⫺ efflux) but in other studies, renal NBCe1, including that of rabbit, is calculated to operate with an apparent 1:3 Na:HCO3 stoichiometry (348, 896, 1085). Indeed, some studies suggest that NBCe1 could fulfill its physiological mission of HCO3⫺ reabsorption only if it operated with a stoichiometry of greater than 1:2 in the PT (381, 1086) (see above). Such a shift in stoichiometry could be achieved by unveiling a cryptic HCO3⫺ cotransport site (e.g., FIGURE 16, A–C versus E–G), or a cryptic H⫹ exchange site (e.g., FIGURE 16, A–C versus H–J). The mechanism(s) that control the apparent change in stoichiometry from 1:2 to 1:3 and vice versa (reviewed in Ref. 349) are unclear but have been suggested to involve a number of factors, such as changes in [Ca2⫹]i (667), changes in the phosphorylation state of the transporter (347), changes in

the direction of transport (750), the presence of an as-yetunidentified binding partner in PT epithelia (344), differences in cell type in which the transporter is being expressed (346), and/or primary culture conditions in the case of proximal tubules (666, 668). III) Substrate specificity. When expressed heterologously, NBCe1 does not require extracellular Cl⫺ to function (138, 339) and, as described above, is inhibited by stilbene disulfonates (140, 611, 853). The Km of the transporter for Na⫹ is ⬃20 –30 mM (634, 822, 853). Rat NBCe1 expressed in Xenopus oocytes mediates a small amount of Li/HCO3 cotransport (estimated at ⬃3% of Na/HCO3 cotransport) but does not mediate K/HCO3 cotransport (853). We estimate that human NBCe1, as expressed in oocytes, can support ⬃10% Li/HCO3 cotransport compared with Na/ HCO3 cotransport (Lee, Boron, and Parker, unpublished data) but ⬃25% when expressed in a kidney cell line (39). Similarly, NBCe1 assessed in basolateral membrane vesicles from rabbit PT does not exhibit a strong Na⫹/Li⫹ selectivity (897). It is unclear whether the poorer Na⫹/Li⫹ selectivity in renal membranes versus oocyte membranes reflects differences in NBCe1 behavior, assay method, or contributions from other endogenous kidney transporters. The Km of NBCe1 for HCO3⫺ is ⬃4 –10 mM (339, 543, 634). Preliminary studies indicate that at least rat NBCe1 expressed in Xenopus oocytes can also transport select anions other than HCO3⫺/CO32⫺, such as NO3– (852). Na⫹coupled, DIDS-sensitive HSO3⫺/SO32⫺ cotransport attributed to NBCe1 has been reported in rabbit PT vesicles (893)

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Na+

F

H+

MARK D. PARKER AND WALTER F. BORON of NBCe1-B, -C, and -E in diverse cell types (FIGURE 17C).33 Translation of these variants begins at exon 2 (initiator methionines are marked “M” in FIGURE 17C). The transcription of NBCe1-B from promoter P1 in mouse ameloblast-like LS8 cells is pH-dependent: transcript abundance is increased in acid-incubated cells and decreased in alkaliincubated cells (706, 891). The human P1 region includes a 284 bp, “pH-responsive” sequence that ends 8 bp upstream of the transcriptional start site (891). If this sequence is placed upstream of a reporter gene that has a minimal promoter, the transcription of the reporter in LS8 cells is enhanced when the cells are maintained in media with an acidic pH (pH 6.8 versus pH 7.4; Ref. 891). The action of the “pH-responsive” enhancer requires DNA elements that contain consensus binding sites for NF-␬B and p53 (891).

and in oocytes injected with rabbit kidney RNA (822), but not in oocytes expressing human (339) or rabbit (Lee, Boron, and Parker, unpublished data) NBCe1, suggesting that the observations from renal preparations could be complicated by the presence of other anion transporters, such as Slc26a1 (FIGURE 1). Finally, according to one report, NBCe1-A, at least at high extracellular pH, might mediate a small degree of OH⫺ transport (39). D) THE SLC4A4 GENE.

33 Only the ORFs, and not the 5= UTRs, have not been reported for NBCe1-C, -D, and -E so the presence of exon 1 has not been demonstrated in these transcripts. We cannot rule out the possibility that another promoter is present between exons 1 and 2.

The SLC4A4 gene has two distinct promoters (P1 and P2 in FIGURE 17B, see Ref. 9). The first promoter, P1, is located upstream of noncoding exon 1 and promotes transcription

A

Locus 4q21 50kb

DCK

B

SLC4A4

Gene structure 10 kb

P1

1

C

GC

2

P2

3

4

9

24

Transcript variation P1

P2 M

4

NBCe1-A NBCe1-B

5

6

7

23

24

* 25

26

24

* 25

26

24

25 * 25

26

24

* 25

26

M

1

2

3

4a

5

6

7

23

3

4a

5

6

7

23

5

6a

7

23

M

NBCe1-C

1

M

4

NBCe1-D NBCe1-E

2

M

1

2

3

4a

5

6a

7

23

* 26

FIGURE 17. SLC4A4 gene structure and NBCe1 transcript variants. Scale diagrams showing the human SLC4A4 gene locus together with the position of neighboring genes (A), the position of promoters (P1 and P2), and the position of exons within SLC4A4 (B). Transcript variants are represented, not to scale, as numbered boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature transcript. “//” denotes that all five transcripts include exons 7–23. Exons that include the initiator ATG codon (“M”) and termination codon (“*”) are marked for each transcript. Sequences that are derived from part of a larger exon sequence are labeled with an “a” (e.g., exon 4a is a subdivision of exon 4). Colored exons, or parts of exons, correspond to the protein regions that each encodes, which are identically colored in FIGURE 18. Uncolored exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line are predicted, but not demonstrated, to be included in the mRNA.

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The human NBCe1 gene maps to chromosomal locus 4q21 (6) and has at least 26 exons that encompass ⬃390 kb of genomic DNA. As shown in FIGURE 17A, the upstream neighbor of SLC4A4 is DCK (deoxycytidine kinase) and the downstream neighbor of SLC4A4, transcribed from the opposite DNA strand, is GC (groupspecific complement, vitamin D binding protein).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

The second promoter, P2, is located upstream of exon 4 (FIGURE 17B) and promotes transcription of NBCe1-A, and likely also NBCe1-D (FIGURE 17C). Translation of NBCe1A/D begins in exon 4 (initiator methionines are marked “M” in FIGURE 17C). The P2 promoter is very active in renal PT cells.

The 85-amino acid Nt appendage (FIGURE 18/blue module) common to NBCe1-B, -C, and -E (encoded by exons 2 and 3 and beginning with the amino acid sequence “MEDE-”) includes an autoinhibitory domain (AID) that inhibits NBCe1 activity (634). The AID also includes binding determinants (IBD) for the NBCe1 activating protein IRBIT (881).

E) STRUCTURAL FEATURES AND VARIANTS OF NBCe1.

B) Cassette I. In NBCe1-D/E, the excision of a 27 nt region, homologous to cassette I of NBCn1, arises due to the use of a cryptic splice site within exon 6 of the gene (see FIGURE 17C and Ref. 599). Omission of cassette I (FIGURE 18/purple module) is predicted to shorten the Nt loop region (see FIGURE 15) by nine residues (loss of “RMFSNPDNG” in mouse NBCe1). The effect of losing cassette I is unknown, although cassette I does contain a consensus casein kinase II phosphorylation site (599), indicating a regulatory role. Transcripts lacking cassette I appear to be widely distributed but only account for a small fraction of the pool of total NBCe1 transcripts that had previously been identified as NBCe1-A/B in any given organ (599). Omission of cassette I from NBCe1C-like transcripts has not been reported.

I) Sources of variation in coding sequence among NBCe1 variants. A) Alternative Nt appendages (“MSTE-” versus “MEDE-”). The mechanisms that result in the production of two alternative NBCe1 Nt appendages (FIGURE 18/blue versus red modules) are shown in FIGURE 17C. The 41amino acid Nt appendage (FIGURE 18/red module) common to NBCe1-A and NBCe1-D (encoded by exon 4 and beginning with the amino acid sequence “MSTE-” ) includes an autostimulatory domain (ASD) that enhances NBCe1 activity (634).

C) Alternative Ct (“-HTSC” versus “-ETTL”). Alternative splicing of exon 24 (the length of which is not a multiple of 3 nt) in NBCe1 transcripts determines the reading frame in which exon 25 is translated, impacting the remainder of the Ct sequence. In NBCe1-A/B/D/E transcripts, exon 24 (which encodes a 32-amino acid sequence) is spliced to exon 25 (which encodes a 14-amino acid sequence, followed by a termination codon) produc-

TMD

Ct

ASD NBCe1-A

AID

6–9

10–14

NBCe1-C

85

46

1,035

46

1,079

9

61

41

85

PDZ

85

NBCe1-E

1–5

41

NBCe1-B

NBCe1-D

C as se tte

I

Nt

1,094

46

1,026

46

1,070

100 aa

FIGURE 18. NBCe1 protein variants. Scale diagram of protein variants that are encoded by the transcripts represented in FIGURE 17C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino acids and colored to denote their genetic origin as shown in FIGURE 17C. NBCe1-A and NBCe1-D include an autostimulatory domain (ASD), whereas all other variants include an autoinhibitory domain (AID). NBCe1-C terminates with a PDZ-domain binding sequence. A color-matched protein sequence alignment of the variants is provided in Appendix V.

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The five distinct Slc4a4-encoded transcripts (FIGURE 17C) encode protein products NBCe1-A through NBCe1-E (FIGURE 18). Variants differ in the inclusion of one of two distinct Nt appendages, the exclusion of a 9-amino acid cassette I within the Nt domain, and the choice of one of two distinct Ct appendages. Below, we consider in detail the mechanisms that generate this diversity, the similarities and differences among the variants and, anticipating the next section of this review, briefly outline the distribution of each variant. The splicing of NBCe1 along with that of other renal transporters has been reviewed in Ref. 310.

MARK D. PARKER AND WALTER F. BORON ing a 46-amino acid Ct appendage (FIGURE 18/green module) that terminates with the sequence “-HTSC”. The remainder of exon 25 and all of terminal-exon 26 of NBCe1-A/B/D/E comprise the 3=-UTR (FIGURE 17C).

II) Cloned NBCe1 variants that are demonstrated or likely to exhibit NCBT activity. A representation of the five variants NBCe1-A through NBCe1-E is shown in FIGURE 18, and the composition of each is described below. Also listed here are the major anatomical locations from which each variant has been cloned as a full-length cDNA (the only reliable demonstration of the presence of each in any preparation). Distribution of subsets of NBCe1 variants (such as might be determined using an antibody that recognizes the common Ct of NBCe1-A/B/D/E) are discussed separately in section “Distribution of NBCe1” below. GenBank protein accession numbers for the variants discussed in this section are provided in Appendix IV. A) NBCe1-A (NCBT activity demonstrated). This predominantly renal variant of NBCe1 (64, 138, 806) has a predicted nonglycosylated molecular mass of 116 kDa (190). NBCe1-A includes 1) the 41-amino acid “MSTE-” Nt sequence that includes an ASD, 2) cassette I, and 3) the 46amino acid “-HTSC” Ct sequence. Due to the presence of the ASD, NBCe1-A has a greater per-molecule activity than either NBCe1-B or NBCe1-C (634). NBCe1-A has also been cloned from testis, epididymis, and ovary (599). B) NBCe1-B (NCBT activity demonstrated). This widely expressed splice form of NBCe1 (6, 192) includes 1) the 85-amino acid “MEDE-” Nt sequence that contains an AID and an IRBIT-binding sequence, 2) cassette I, and 3) the 46-amino acid “-HTSC” Ct sequence. Due to the presence of the AID, NBCe1-B has a lower per-molecule activity than NBCe1-A and a similar per-molecule activity to NBCe1-C (634). Apart from the pancreas, where NBCe1-B transcripts are most abundant, NBCe1-B has been cloned from the

850

C) NBCe1-C (NCBT activity demonstrated). This predominantly brain-expressed variant (79) includes 1) the 85amino acid “MEDE-” Nt sequence that constitutes an AID and an IRBIT-binding sequence, 2) cassette I, and 3) the 61-amino acid “-ETTL” Ct sequence. NBCe1-C is uniquely distinguished by the presence of the 61-amino acid Ct as it is the sole variant that includes this Ct. NBCe1-C has also been cloned from murine epididymis and testis (599) and human heart. Due to the presence of the AID, NBCe1-C has a lower per-molecule activity than NBCe1-A and a similar per-molecule activity to NBCe1-B (634). D) NBCe1-D (NCBT activity untested). NBCe1-D is identical to NBCe1-A except for the absence of cassette I. Transcripts lacking cassette I appear to be widely distributed but only account for a small fraction of the pool of total NBCe1 transcripts that, until now, had been identified as NBCe1-A in any given preparation (599). Full-length NBCe1-D cDNA has been cloned from murine epididymis (599). We regard NBCe1-D as likely to have NCBT activity because NBCn1-H, which lacks the homologous cassette I, has NBCn1 activity. E) NBCe1-E (NCBT activity untested). This variant is identical to NBCe1-B in its coding sequence except for the absence of cassette I (599). NBCe1-E transcripts only account for a small fraction of the pool of total NBCe1 transcripts that until now had been identified as NBCe1-B in any given preparation (599). Full-length NBCe1-E cDNA has been cloned from murine ovary, uterus, and epididymis (599). We regard NBCe1-E as likely to have NCBT activity because NBCn1-H, which lacks the homologous cassette I, has NBCn1 activity. III) Predicted NBCe1 variants. NBCe1 variants that include the 61-amino acid Ct of NBCe1-C and that also 1) include the ASD of NBCe1-A or 2) lack cassette I have not been reported. Possibly the splice machinery that excises exon 24 is absent from the pool of cell types that promote NBCe1-A transcription. A lab-created, chimeric NBCe1 that includes the Nt of NBCe1-A and the Ct of NBCe1-C is reported to exhibit an activity that is slightly greater than NBCe1-A (634), consistent with the mildly stimulatory effect of the NBCe1-C Ct in the absence of an Nt AID (634). IV) Other NBCe1 variants. We are not aware of any cloned or predicted NBCe1 variants besides those mentioned above. F) DISTRIBUTION OF NBCe1.

The major organs most often associated with NBCe1 expression are the pancreas and kidney, although NBCe1 is also abundant in many other organs.

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In NBCe1-C transcripts, exon 24 is omitted (FIGURE 17C). Due to the resulting frame shift, exon 25 now encodes a 27-amino acid sequence and the terminal exon 26 encodes a 34-amino acid sequence followed by a termination codon and the 3=-UTR. Thus it is that, in NBCe1-C, exons 25–26 encode a 61-amino acid Ct appendage (FIGURE 18/orange module) that terminates with the sequence “-ETTL”. The consequences of alternative Ct choice are unclear, but “-ETTL” is a PDZ-binding domain interacting sequence (79) and deletion of either the 46-amino acid or the 61amino acid Ct sequences results in reduced NBCe1 accumulation in the plasma membrane (276, 634). In the absence of the Nt autoinhibitory domain, NBCe1-C has a greater activity than NBCe1-B, as if the 61-amino acid Ct appendage is stimulatory, or the 46-amino acid appendage is inhibitory, in the absence of the Nt AID (634).

brain (79), cornea (922), heart (192), parotid salivary gland (508, 710), ileum (64), and from diverse tissues within the male and female reproductive tracts (599).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

The distribution of NBCe134 in specific organ systems is discussed below. The distribution of NBCe1 is summarized and compared with that of other NCBTs in TABLE 5. In instances where a detection method would not distinguish between two variants, for example, use of an antibody against the common Nt of NBCe1-B and NBCe1-C, we refer to NBCe1-B/C. If it is unknown which variant is being discussed, or in instance of organs in which there appears to be no obvious bias in variant expression, we refer to NBCe1 without a variant designation.

34 Antibodies and most PCR probes used in these studies would not be able to differentiate NBCe1-A from NBCe1-D, or NBCe1-B from NBCe1-E, but NBCe1-D and NBCe1-E appear to only account for a minor fraction of total NBCe1 product and are not considered here in the discussion of reports of NBCe1-A and NBCe1-B. 35 NBCe1-A transcripts in mouse brain are nearly 50% as abundant as those encoding NBCe1-B/C (E. Roussa, personal communication).

As determined by in situ hybridization or immunohistochemistry, NBCe1-B and NBCe1-C are present throughout the rat brain but exhibit particularly robust expression in the dentate gyrus of the hippocampus, cerebellum, olfactory bulb, and piriform cortex (318, 624, 843), and in the brain stem/diencephalon region (260, 1060). In general, NBCe1-C transcripts appear to outnumber those encoding NBCe1-B (624), although the ratio of NBCe1-A/B (likely NBCe1-B) to NBCe1-C protein is greater in cerebellum compared with other brain regions (261, 1060). In rats, expression of NBCe1 transcripts (318) and protein (260) is not detected in the brain until birth, whereupon NBCe1 levels increase gradually until an age of 4 wk (260).

36 The immunostaining of NBCe1-A protein throughout the mouse brain must be interpreted with some caution: 1) the preimmune serum from the rabbit used to generate the anti-NBCe1-A antibody diffusely labeled tubules in the rat renal cortex (see Fig. 3C of Ref. 817); and 2) no preimmune controls are presented for the brain sections (796).

Table 5. Sites of NCBT expression

Central nervous system Sensory organs Peripheral nervous system Respiratory system Circulatory system Musculoskeletal system Upper digestive system Lower digestive system

NBCe1

NBCe2

NBCn1

NDCBE

NBCn2

Widespread, neurons and astrocytes Eye Trigeminal ganglion

Blood-brain barrier and elsewhere Eye Trigeminal ganglion

Widespread, neurons

Widespread, neurons

Widespread, mainly neurons Eye, ear

Nose and elsewhere

Lung

Trachea and lung

Trachea and lung

Vasculature

Heart

Heart

Osteoclasts, skeletal muscle Widespread

Skeletal muscle

Skeletal muscle

Cardiac myocytes and Heart elsewhere Skeletal muscle Skeletal muscle Widespread

Stomach

Widespread, abundant in pancreas

Widespread, abundant in liver

Widespread

Widespread

Widespread

Spleen and leukocytes Thyroid Kidney Placenta and testes

Spleen and macrophages

Widespread

Spleen

Widespread Kidney Testes and elsewhere

Pituitary gland Kidney Testes

Lymphatic system Endocrine system Urinary system Reproductive system

Ear Trigeminal ganglion

Thyroid and pancreas Kidney Widespread

Bladder and kidney Widespread

Stomach

A more complete and detailed examination of each NCBT distribution is provided in text. A distribution of NCBT expression based on the origins of corresponding expressed-sequence-tags is provided in Appendix VI.

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I) Central nervous system. A) General. The distribution of NBCe1 variants in the central nervous system was recently reviewed by Majumdar and Bevensee (623). Assessed by PCR, NBCe1-B/C transcripts are abundant in rat brain, whereas NBCe1-A is much less abundant (318). By immunoblot, NBCe1-C is abundant in rat brain versus kidney, whereas NBCe1-A/B expression in brain is negligible compared with kidney (79). In mouse brain (796), NBCe1-A can be identified by quantitative PCR,35 and the protein is

reported to be widespread by immunohistochemistry.36 However, in rat and human brain, northern blot and in situ hybridization studies using NBCe1 variant-specific probes indicate that expression of NBCe1-A transcripts is insubstantial (6, 318, 624). Thus it would appear that the predominant NBCe1 variants in mammalian brain are NBCe1-B and NBCe1-C.

MARK D. PARKER AND WALTER F. BORON B) Neurons versus glia. In primary cultures from cerebral cortex, NBCe1-B protein is expressed mainly in astrocytes, whereas NBCe1-C is expressed mostly in neurons (79). However, the expression pattern appears to be just the opposite in situ (624), where immunohistochemistry and immuno-gold labeling reveals NBCe1-A/B (likely NBCe1-B) inside neurons, and reveals NBCe1-C on the plasma membrane of astrocytes. A study on mouse or rat brain oligodendrocytes demonstrates NBCe1-A/B (likely NBCe1-B) immunoreactivity in the dendrites of these cells (800).

D) Blood-brain barrier. NBCe1 protein has been detected in basolateral membranes of choroid plexus epithelia (843). Transcripts also are detected in the outer meningeal layer (843). II) Sensory Organs. A) Eye. NBCe1-B is often described as the major NBCe1 variant expressed in the eye, although most evidence relies on molecular tools that do not discriminate between NBCe1-B and NBCe1-C. To our knowledge, an antibody specific for NBCe1-C has never been used to examine the distribution of NBCe1 in the eye. In one study on human corneal endothelium, a primer pair that should amplify both NBCe1-B and NBCe1-C yielded one fulllength cDNA clone, which corresponds to full-length NBCe1-B (922), but this does not exclude the presence of NBCe1-C. In ciliary body, an antibody that recognizes both NBCe1-A and NBCe1-B exhibits robust immunoreactivity (1013), even though NBCe1-A is not abundantly expressed in the eye (see below). Taken together, these data are consistent with the hypothesis that NBCe1-B is the dominant NBCe1 variant in the eye. NBCe1-B/C cDNA and protein are detected in a variety of ocular tissues, namely the surface and wing cells, but not the stroma, of the conjunctiva (94);37 the keratocytes of the corneal stroma (94); the endothelial cells of the cornea (94, 248, 593, 922, 923, 989, 990), predominantly at the basolateral membrane (94, 248, 922, 923, 989; see cartoon in FIGURE 19); the trabecular meshwork in the anterior chamber (989), responsible for draining aqueous humor; the pigmented epithelium of the ciliary body (94, 989) in the posterior chamber, specifically at the basolateral membrane of the pigmented cells (94). Data conflict concerning the presence of NBCe1 in the non-pigmented epithelia of the ciliary body (94, 868, 989), which is responsible for secretion of the aque37 In a separate study, Turner and co-workers were unable to demonstrate NBCe1-A/B immunoreactivity in the conjunctival epithelium of rats and pigs because their immunohistochemical studies were hampered by “discernable nonspecific labeling” (985).

852

A small amount of NBCe1-A cDNA has been detected by PCR in a human corneal endothelial cell line, but this is swamped by a greater population of NBCe1-B/C cDNAs (990). On the other hand, the cornea per se is reported to be negative for NBCe1-A cDNA in cattle (923) and humans (922), and corneal endothelium is negative for NBCe1-A protein in rat (94). A report of NBCe1-A expression in rat ciliary body was based on an antibody raised against an epitope common to NBCe1-A and NBCe1-B (1013), and a report of NBCe1-A expression in porcine nonpigmented ciliary epithelial cDNA depended on a primer pair that does not distinguish among NBCe1 variants (868). Indeed, an antibody study by Bok et al. (94) found only NBCe1-B, and not NBCe1-A, expression in the ciliary body. An immunohistochemical study by the same workers did detect NBCe1-A in the basal epithelium of rat conjunctiva (94). Only one study reports appreciable NBCe1-A protein expression elsewhere in the rat eye: using immunohistochemistry, Usui and co-workers found NBCe1-A protein together with NBCe1-B protein in the ciliary body, lens, and cornea of the rat eye (990). However, the NBCe1-A immunoreactivity was diffuse, in contrast to the clear membrane localization of NBCe1-B immunoreactivity in the study of Bok and co-workers. Despite the relatively low abundance of NBCe1-A in the eye, it is interesting to note that an individual with the mutation Q29X, predicted to specifically eliminate NBCe1-A (FIGURE 25 AND TABLE 6), has bilateral glaucoma (412). Thus either NBCe1-A is expressed in tissues involved in regulating anterior-chamber volume (e.g., nonpigmented epithelium of the ciliary body, trabecular meshwork), presumably early in development, or the ocular phenotype is secondary to the whole body acidosis caused by NBCe1-A deficit in the kidney. III) Peripheral nervous system. A) Trigeminal ganglion. NBCe1-B/C, but not NBCe1-A, transcripts are detected by reverse transcription polymerase chain reaction (RT-PCR) in preparations of rat trigeminal ganglion neurons (408). IV) Respiratory system. A) Nose. In human nasal mucosa, NBCe1-A, but not NBCe1-B/C, transcripts are detected in the epithelia and submuscosal gland cells of the inferior turbinate mucosa, and in the superficial epithelia of nasal polyps (558). B) Lungs. NBCe1-B/C immunoreactivity is detected in preparations of basolateral membrane proteins of the Calu

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C) Spinal cord. NBCe1 transcripts are detected in the developing rat spinal cord from embryonic day 19 (318), and NBCe1 protein has been noted in the spinal cord (both white and gray matter; Ref. 843), according with a substantial presence of NBCe1-B/C transcripts in spinal cord mRNA (6).

ous humor; the epithelium of the lens, in both apical and basolateral membranes, and a human lens anterior epithelium cell line (94, 875, 989); and the retina (30) including specifically the apical microvilli and end feet of Müller glial cells (94), the apical membrane of retinal pigment epithelial cells (11, 94), and the choriocapillaris (1079).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Aqueous humor

Tight junction

Stroma

Na+ NBCe1 Na+

CFTR

2 HCO3–

Cl–

Na-K pump 3 Na+ 2 K+

HCO3–

Stroma

NHE H+

Na+ Lens NKCC1

Corneal endothelium

Na+ CA

K+ Aqueous Humor

2 Cl– CO2

KCNQ1 H2O

K+

Corneal endothelium FIGURE 19. Role of NBCe1 in the cornea. The corneal endothelium reabsorbs fluid from the collagen matrix that constitutes the stroma, preventing corneal edema (see cartoon of eye). NBCe1-B in the basolateral membrane supports transepithelial anion secretion. Note the similarities between this pathway and the mechanism of NaCl secretion in dogfish salt glands (FIGURE 11).

cell line, which is derived from pulmonary airway submucosal-gland serous cells (515). V) Circulatory system. A) Heart. Most northern blot analyses of human mRNAs and qPCR experiments indicate that NBCe1 is expressed in the heart, although at a lower abundance than in kidney or pancreas (6, 31, 140, 192, 480, 684, 831). Full-length NBCe1-B has been cloned from human heart cDNA (192), and an antibody directed against the third extracellular loop of NBCe1 immunoreacts with protein in rat and human ventricular myocardial cells (481). NBCe1-A/B immunoreactivity, likely representing NBCe1-B, is present in the left and right ventricles as well as in the interventricular septum of rat heart (831). A preliminary immunocytochemical study of rat ventricular myocytes suggests that NBCe1 protein is located in the traverse (T) tubules, in contrast to the predominantly surface-sarcolemmal distribution of NHE1 (311). B) Capillaries. In the testes of rats, NBCe1 immunoreactivity is detected in capillary-lining endothelial cells (445).

VI) Musculoskeletal system. A) Skeletal muscle. NBCe1 immunoreactivity is detected in skeletal muscle homogenates from humans and rats (518) and has been detected in soleus and extensor digitorum longus (i.e., calf) muscles of rats (964). Immunohistochemistry of rat muscle suggests that NBCe1 is located in the sarcolemmal membrane and perhaps also, the authors of the study suggest, in T tubules (518). VII) Upper digestive system. A) Enamel organ. Ameloblasts promote enamel deposition on developing teeth and NBCe1-B, but not NBCe1-A, transcripts are detected in preparations of microdissected ameloblasts from mice and humans (538, 1099).38 NBCe1 transcripts are more abundant in mature than secretory ameloblasts (539, 540, 1099). Immunohistochemistry appears to demonstrate a

38 The report of human NBCe1-A, and not NBCe1-B, expression in microdissected human enamel organ (1099) is probably incorrect. The “human NBCe1-A” primer pair used in Ref. 1099 is actually specific to NBCe1-B/C, whereas the “human NBCe1-B” primer pair used in Ref. 1099 is actually specific for NBCe1-A.

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cAMP HCO3–

Cornea

H+

++

MARK D. PARKER AND WALTER F. BORON basolateral distribution of NBCe1-A/B protein in mouse ameloblasts (538, 706), with an additional presence in the adjoining stratum intermedium of the papillary cell layer (538). However, high-resolution images presented in a study of mouse dentition disclose NBCe1-A/B immunoreactivity only in the stratum intermedium, with no NBCe1 expression in the ameloblasts themselves (456), as depicted in the cartoon in FIGURE 20.

strong basolateral NBCe1 immunoreactivity is present in the parotid acini of humans (using an anti-NBCe1-B/C antibody; Ref. 710) and rats (anti-NBCe1; Ref. 818) as well as in a rat parotid acinar cell line (anti-NBCe1-A/B; Ref. 740). Taken together, these data suggest that NBCe1-B is the major NBCe1 variant expressed in parotid acini. To our knowledge, the presence of NBCe1-C in salivary glands has not been examined.

Three factors could underlie the apparent discrepancy among the above studies: 1) it is difficult to resolve the ameloblast basolateral membrane from the membranes of abutting papillary cells in the stratum intermedium, 2) the studies were performed in different species, and 3) the studies employed different antibodies.

Apart from acinar cells, NBCe1 immunoreactivity is also evident in the basolateral membranes of striated and main duct cells of rat parotid glands (818). In duct cells, NBCe1 would act in parallel with NBCn1 (see cartoon in FIGURE 21B).

Interstitial space

NBCe1-B

Na+ Na-K pump 2 HCO3–

2 K+ Papillary cell 3 Na+

Na+ HCO3–

NBCn1 H+

Gap junction

Tight junction

+ NHE +

Basal

Lateral

Ameloblast

Na+

HCO3–

CO2

cAMP

CA H+

H2O Cl–

Cl– Apical Enamel surface compartment

HCO3– AE2 Slc26a4?

CFTR Cl–

HCO3–

FIGURE 20. Role of NBCe1 in the enamel organ. Apatite formation in the enamel compartment generates ⫺ ⫺ secreted from mature ameloblasts. NBCe1 mediates HCO3 influx in papillary H⫹ that are neutralized by HCO3 ⫺ ⫺ ⫺ cells, and the HCO3 is transferred to ameloblasts via connecting gap junctions. Transported HCO3 and HCO3 generated within ameloblasts by CA is secreting into the enamel compartment via lateral AE2 and perhaps apical pendrin (Slc26a4). The presence of pendrin in the apical membrane of ameloblasts is controversial (124, 125, 297). The extracellular face of AE2 is exposed to the enamel compartment by a rearrangement of tight junctions (456).

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B) Salivary gland. In parotid salivary glands, the acinar cells are a site of NBCe1 expression (see cartoon in FIGURE 21A). Only NBCe1-B/C, and not NBCe1-A, is detected by PCR of mouse and bovine parotid cDNA (490, 1065). NBCe1-B was cloned from these cells in guinea pigs (508). Moreover,

Concerning the sublingual and submandibular glands, NBCe1 transcripts are absent from cDNA prepared from the sublingual salivary glands of mice (490), but are detected in the submandibular glands of guinea pigs (508). Furthermore, two studies describe NBCe1 immunoreactivity in the basolateral membranes of rodent submandibular gland duct cells (615, 818).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

A

B

Lumen Na+ H2O

Fluid secretion TMEM16A

CFTR

Acinus

Cl–

Slc26a6

Slc26a3

Duct

–

HCO3

Cl– H+

H2O CAII HCO3–

CO2

cAMP Na+ 2 HCO3–

NHE1

Na+ 2 HCO3– Na+ HCO3–

Na+

++

Na+ K+ 2 Cl–

++

Cl–

H+

CO2

K+

HCO3– AE2

NBCe1-B

H+ NKCC1

Salivary acinar cell

NHE1

NBCe1-B

NBCn1

Salivary duct cell

FIGURE 21. Role of NCBTs in exocrine glands. The inset in A displays a generic acinus (acinar epithelia, blue) and duct (duct epithelia, yellow) for an exocrine gland such as the salivary gland or pancreas. NBCe1 activity regulates intracellular pH and could support transepithelial fluid and ion secretion by salivary gland ⫺ acinar cells (A). NBCe1 and NBCn1 support transepithelial HCO3 secretion by salivary gland duct cells (B), ⫺ -rich saliva. The presence of AE2 in the duct cells of salivary glands may contributing to formation of a HCO3 be species specific [present in humans (998) but not in rats (818)]. Similarly, the presence of NKCC1 in duct cells is not reported in all species (e.g., absent from mice in Ref. 279). The Na pump has been omitted from both cell types for clarity. A similar mechanism for fluid secretion operates in pancreatic acini and ducts.

C) Esophagus. NBCe1-A/B immunoreactivity is present in the basolateral membranes of acinar and duct cells of esophageal submucosal glands (3, 4). NBCe1 is also detected in enzyme-secreting serous cells, but here the polarity of NBCe1 distribution is not evident (3, 4). D) Stomach. NBCe1-B/C transcripts are present in stomach preparations from rabbits (427), guinea pigs (508), and humans (6). Northern blots and qPCR of rabbit gastric mucosal cell preparations suggest that NBCe1 is more abundant in mucous cells than chief or parietal cells (814). NBCe1 transcripts are also present in a cell line derived from rat gastric mucosa (369). VIII) Lower digestive system. A) Intestines. At the level of mRNA or cDNA, NBCe1-B (or NBCe1-B/C) is widely expressed in the lower digestive tract. Full-length NBCe1-B has been cloned from rabbit duodenum (427). Intestinal expression of NBCe1-B/C transcripts has also been demonstrated in 1) rabbit colonic mucosa, with lower levels of expression in the ileum (427); 2) mouse duodenum (753)

and colon (1087) [in the mouse proximal colon, in situ hybridization detects NBCe1 transcripts only in crypt epithelia (55)]; 3) rat small intestine and colon (318) [along the rat distal colon, NBCe1 transcripts are more numerous in the last quarter (furthest from the lymph node), than the first quarter (closest to the lymph node; see Ref. 1059)]; 4) guinea pig small intestine and proximal colon (508); 5) opossum ileum (64); and 6) human colon (6). Aside from NBCe1-B/C, a small population of NBCe1-A transcripts is detected in the ileum and colon of rabbits (427) as well as in the duodenum (753) and colon (482) of mice, but NBCe1-A is not present at appreciable levels in the ileum of opossums (64). NBCe1-A is reportedly the predominant form of NBCe1 expressed in the human cancer cell line HT29, although evidence for the assignment is not provided (642). At the level of protein, NBCe1 immunoreactivity is detected in mouse duodenum in the basolateral membranes of enterocytes (see FIGURE 22 and Ref. 753). In the enterocytes of

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cAMP

Interstitium

Na+ H2O

Na+

Cl–

CAII

Na+

HCO3

H+

H2O HCO3–

ENaC –

MARK D. PARKER AND WALTER F. BORON

Lumen

Tight junction

Interstitium

Na+

Na+

NHE3

NBCe1-B

H+

2 HCO3–

cAMP

Na+ NBCn1 HCO3–

Cl–

CFTR

Na+ K+ Slc26a6

H+

Cl–

2

NKCC1

Cl–

HCO3– Slc26a3

K+

Cl–

HCO3–

CA

Na+ cAMP

CO2 H2O

++

H+

Cl– HCO3–

NHE

AE2

Duodenal villar enterocyte FIGURE 22. Role of NCBTs in intestine. Shown is a duodenal villus enterocyte. The mechanism of transep⫺ ithelial fluid and HCO3 secretion is very similar to that shown in FIGURE 21 for salivary glands. The Na pump is omitted for clarity.

rat proximal duodenum, NBCe1 immunoreactivity is strongest in villar enterocytes and decreased in abundance closer to the crypts, such that NBCe1 immunoreactivity is not detectable in goblet cells (430). A similar distribution is detected in opossum ileum (64). NBCe1 immunoreactivity is also evident in the enterocytes of the proximal jejunum (villar and crypt enterocytes), ileum and proximal, but not distal, colon (430). NBCe1 immunoreactivity has also been detected in the basolateral membranes of brush cells from rat cecum (696). B) Liver. Northern blots indicate that the liver may be an additional, albeit minor, site of NBCe1 expression (6, 806). In rat bile duct, basolateral NBCe1 immunoreactivity is detected in brush cells that are hypothesized to secrete HCO3⫺ (695). C) Pancreas. At the mRNA level, the pancreas is the single most abundant site of expression of NBCe1, specifically NBCe1-B (6). The role of NBCe1 in this organ is also reviewed in Reference 906. NBCe1-B has been cloned from human pancreatic cDNA (6). Taken together, in situ hy-

856

bridization and immunohistochemical data demonstrate that pancreatic NBCe1 is expressed in the acinar and duct cells. NBCe1 is also expressed in insulin-secreting ␤ cells in the islets. In the acinar cells of mice, NBCe1-B transcripts have been detected by in situ hybridization (6) and NBCe1-B protein is located in the basolateral membrane of rat acinar cells (817, 836, 962). However, no NBCe1-B immunoreactivity is detected in human pancreatic acini (626, 836). In the duct cells of the human pancreas, one study, using antibodies to an Nt epitope or a Ct epitope that are common to all NBCe1 variants, demonstrated that NBCe1 colocalizes with Na-K pump (basolateral) but not with CFTR (apical) (626). In other studies investigators variously report NBCe1-B immunoreactivity in the duct cells of both human and rats as apical and/or basolateral, or as different among duct types and even among cells in the same duct (94, 817, 836, 962). In some cases this confusion may arise from quality-control issues surrounding the use of antibod-

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HCO3–

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

ies raised to be specific to NBCe1-B.39 NBCe1-B is also expressed in the pancreatic duct cell lines CAPAN-1 (883) and mPEC1 (344), as well as in the cystic fibrotic pancreatic duct cell line CFPAN-1 (883). Physiological data support an exclusive presence of NCBT activity in the basolateral membranes of duct cells (422, 883, 1096).

IX) Lymphatic and immune systems. As far as we are aware, there are no reports of substantial NBCe1 expression in the lymphatic or immune systems. An NCBI-curated database reports a small number of human NBCe1 ESTs derived from bone marrow and spleen (Appendix VI). However, NBCe1 transcripts are noted as undetectable by northern blot of mouse spleen RNA (313).

XI) Urinary system. A) Kidney. The kidney is the major site of expression for the NBCe1-A transcript (6). Renal NBCe1-A transcripts have been cloned from many species including humans (138) and rats (140, 806). NBCe1-A protein is expressed in the kidney cortex, specifically in the basolateral membranes of the S1 (i.e., just distal to Bowman’s capsule) and early S2 PT segments in humans, rabbits, and rats (7, 273, 632, 829, 844, 1024, 1064), as depicted in the cartoon in FIGURE 23. The segmental distribution of NBCe1 along the nephron significantly overlaps with that of protein 4.1B in the S1 and S2 tubules (958), and with the Na/glucose cotransporter SGLT1 in the S2 tubule (806). A lesser amount of NBCe1 mRNA expression is detected in the S3 proximal tubule segments of rabbits (7), as expected from a tubule segment in which HCO3⫺ reabsorption is less than for the S1 and S2 segments (7). Indeed, NBCe1 protein is totally absent from the S3 segment of rats (632). Traces of NBCe1 transcript expression have also been detected in the renal medulla of rats (140) and in a mouse cell line from the inner medullary collecting duct (35).

X) Endocrine system. A) Thyroid. NBCe1 transcripts are detected in extracts prepared from human thyroid (309, 486). B) Pancreas. NBCe1-B transcripts are not detected in pancreatic islet cells of mice (6). However, NBCe1-A and NBCe1-B transcripts are detected in the pancreatic islet cells of rats, although the immunoreactivity of anti-NBCe1-A and -B/C antibodies are not robust at the level of western blots (901). In immunohistochemical studies on rat using those same antibodies, NBCe1-B immunoreactivity is detected in the ␤ cells that secrete insulin but not in the ␣ cells that secrete glucagon (901). Moreover, NBCe1-A/B immunoreactivity is detected in insulin-positive cells of pancreatic samples isolated from human cadavers (365). NBCe1-B immunoreactivity is also expressed in the insulin-secreting cell

Lumen

Some caution must be exercised when interpreting these studies, as the antibodies raised against epitopes in the common Nt of NBCe1-B/C appear to be troublesome. For example, one NBCe1-B antibody (796, 817) 1) exhibits more robust immunoreactivity with renal protein extracts than with pancreatic protein extracts, opposite to the distribution of NBCe1-B transcripts; 2) immunoreacts with a number of other proteins in pancreatic extracts, such that full-length NBCe1-B is a minor target for this antibody in the pancreas; and 3) exhibits a staining pattern in renal sections that is not different from the nonspecific staining produced using preimmune serum from a rabbit used in the same study (See Fig. 3, C versus D, in Ref. 817). Another NBCe1-B antibody, originally reported in Ref. 989, as expected, does not immunoreact with protein in human kidney extracts but does immunoreact with a rat kidney protein (273).

Interstitial fluid

NHE1

NHE3 Na+

Na+ H+ ++

cAMP

H+

cAMP

++

H+

H+ NBCe1-A

HCO3

HCO3–

H-pump

–

Na+ 3 HCO3–

CAII

CAIV CO2 +

39

Tight junction

H2O +

H2O

3 Na+

CO2 2 K+

AQP1

Na-K pump

Proximal tubule cell

FIGURE 23. Role of NBCe1 in the proximal tubule. H⫹ secreted by proximal tubule (PT) epithelia into the PT lumen can either be titrated by buffers such as phosphate or NH3, in which case they are excreted in the urine or, catalyzed by extracellular CA, they can be ⫺ . CO2 that enters PT epithelia from the lumen, and titrated by HCO3 CO2 that is generated by PT metabolism, is hydrated by CAII into H⫹ ⫺ ⫺ and HCO3 . Reabsorption of HCO3 via NBCe1 drives H⫹ secretion ⫺ and supplies the blood with HCO3 , regulating whole body pH.

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Although, as we have just seen, NBCe1-B is undoubtedly the major, pancreatic NBCe1 variant, it is perhaps not the only one. A pool of NBCe1-A transcripts is detected by PCR from pancreatic cDNA (135, 817, 836, 901) and antibodies raised against an epitope in NBCe1-A immunoreact with a diffuse population of protein in pancreatic duct (817, 836). Considering their relative transcript levels (6), the functional significance of NBCe1-A is likely trivial compared with that of NBCe1-B under basal conditions. To our knowledge, the presence of NBCe1-C in pancreas has not been examined.

line BRIN-BD11 (135). A diffuse staining of NBCe1-A is detected in islet cells (901). In the exocrine pancreas, NBCe1 is also present in acinar and duct cells.

MARK D. PARKER AND WALTER F. BORON NBCe1-A is undoubtedly the major, but perhaps not the only renally expressed NBCe1 variant. A small fraction of NBCe1-B/C transcripts are detected by PCR from renal cDNA (135, 318, 427, 817, 901), and antibodies raised against an epitope in NBCe1-B/C immunoreact with a diffuse subapical population of protein in the rat PT (273, 817). Furthermore, on western blots, an NBCe1-C specific antibody exhibits some immunoreactivity with a rat renal protein extract (79). The expression level of these alternative variants is trivial compared with that of NBCe1-A under basal conditions, but their presence may be of importance during stressed conditions (117).

B) Male. NBCe1 expression is detected in testis (599), epididymis (445, 453, 599, 729), prostate (6, 684), sperm (445), and vas deferens (152, 599). A combination of northern blotting and qPCR data indicate that the prostate is a major site of NBCe1-B/C transcript expression in human males (6, 684). Full-length NBCe1-B has been cloned from human prostate cDNA (GenBank protein accession no. AF053753). NBCe1-A/B immunoreactivity is present in sperm extracts and in the basolateral membranes of apical and principal cells of the epididymis (445).40 In the epididymis, NBCe1-A/B immunoreactivity is most pronounced in the initial segments, growing progressively weaker towards the cauda, a pattern matched by in situ hybridization results using an anti-NBCe1 probe (445). In the testes of rats, NBCe1 immunoreactivity is detected in smooth muscle cells and in capillary-lining endothelial cells (445). The relative abundance of the five NBCe1 variants throughout the mouse reproductive tract is examined in Reference 599. G) PHYSIOLOGICAL ROLES OF NBCe1. Its ability to transport HCO3⫺ across membranes enables NBCe1 to play diverse roles according to its location. In all of the cell types in which NBCe1 is expressed, its action influences pH-sensitive processes within the cell and at the extracellular surface. In polarized epithelia, HCO3⫺ transport can also support HCO3⫺ secretion (i.e., away from the blood) or HCO3⫺ absorption (i.e., toward the blood). Here we first discuss those processes that have general relevance to the function of a number of systems, and then we discuss specialized processes that are specific to certain organs and tissues.

I) General. A) Intracellular pHi regulation. NBCe1 presumably contributes to pHi regulation in every cell in which it is expressed. However, the role played by NBCe1 would depend critically on its stoichiometry. In cultured rat cerebellar (132)

40 A later review by the same group refers to epididymal NBCe1 as NBCe1-A, although the immunohistochemistry by itself is not sufficient to support this specific assignment.

858

A word of caution is that one could easily be fooled by an unanticipated combination of 1) an electroneutral acidbase transporter that requires Na⫹ and HCO3 (e.g., an electroneutral NCBT, or a Na-H exchanger activated by a CO2/HCO3⫺ receptor) and 2) a parallel though unlinked electrogenic process (e.g., a pH-sensitive ion channel). Thus, in reaching the conclusion that an electrogenic Na/ HCO3 cotransporter is responsible for a pHi change, it is important that the investigator verify that the cells do indeed express the transporter, that the rate of pHi change quantitatively matches some measure of electrogenic transport (e.g., a change in Vm but preferably a membrane current), and that the indexes of both transport and electrogenicity have the same ionic and pharmacological properties. A case in point is a report that concluded that, in spinal cord neurons of embryonic rats, an electrogenic NBC contributed to the observed, robust pHi recovery from an acid load (118). The electrical link was a demonstration that an increase in [K⫹]o caused an abrupt pHi increase. However, in salamander PTs, where such a depolarization-induced alkalinization (DIA) was first described, the DIA occurs in the nominal absence of CO2/HCO3⫺ and, in fact, is mediated by electroneutral Na/lactate cotransport across the apical membrane, followed by H/lactate cotransport across the basolateral membrane (884, 885). The spinal cord neuron study did not include an analysis of the Na⫹ or HCO3⫺ dependence of the DIA, nor of its sensitivity to DIDS. Thus one must exercise prudence in interpreting these data. B) Possible role in cell migration. A localized regulatory volume increase (RVI) at the leading edge of lamellipodia in migrating cells is mainly mediated by NHE1 (910). However, on the basis of a residual migratory capability of NHE-deficient MDCK-F cells that is sensitive to the NCBT inhibitor S0859, NBCe1 has been suggested to be capable of making a minor contribution to migration (849). Because 1) S0859 has an untested specificity, 2) NBCe1 transcripts are scarce in these cells, 3) NBCe1 protein expression is undemonstrated in these cells, and 4) other NCBTs aside from NBCe1 may be expressed in these cells, the authors were not able to definitively link NBCe1 activity with cell migration (849).

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XII) Reproductive system. A) Female. NBCe1 transcripts are detected in mouse ovarian, uterine, and vaginal preparations (599, 1027).

and hippocampal (75) astrocytes, an electrogenic Na/HCO3 cotransporter enhances the pHi recovery from an acute intracellular acid load (i.e., the transporter functions as an acid extruder, mediating the uptake of HCO3⫺ equivalents). Thus this transporter, subsequently identified as NBCe1-B in cultured hippocampal astrocytes, must operate with a 1:2 stoichiometry. In renal PTs, NBCe1-A operates with an apparent stoichiometry of 1:3 and thus mediates a net efflux of HCO3⫺ equivalents (i.e., it functions as an acid loader, mediating the efflux of HCO3⫺ equivalents). In these cells, we would expect that NBCe1-A would contribute to the pHi decrease following an acute intracellular alkaline load, although, to our knowledge, this experiment has not been done.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Overexpression of CA IX on the extracellular surface promotes cell migration in MDCK cells (931). Furthermore, NBCe1 and CA IX immunoreactivity colocalize in a hypoxic A549 lung-tumor cell line (254). Thus it has been proposed that CA IX and NBCe1 form a “metabolon” in which CA IX activity (CO2 ⫹ H2O ↔ HCO3⫺ ⫹ H⫹) is promoted by the action of NBCe1 that removes HCO3⫺ from the cell surface (254, 931). The importance of CA IX in tumor pH regulation is reviewed in Ref. 934.

NBCe1 in this regard could be minor compared with that of NHE1.

A study of wound repair in monolayers of a rat gastric epithelial cell line showed that the wound-healing process (i.e., cell migration) could be inhibited by DIDS or by the removal of Na⫹, Cl⫺, and/or HCO3⫺ (369). Although the transport processes responsible for these phenomena remain unidentified, the authors detect both NBCe1 and AE2 transcripts in these cells (369). In summary, although data are consistent with the appealing hypothesis that NBCe1 could support cell migration/ tumor metastasis, the data are not conclusive and the role of

A

Hippocampal neuron

41 Enhanced seizure resistance in a third, NBCe2-null, mouse strain may be an indirect effect of altered CSF composition because NBCe2 is not expressed in neurons.

Alkalinization + Ca2+ release

Neuron firing

+

Decreased [HCO3–]i +

Cl– NDCBE

2 HCO3– Na+

Acidification

Alkalinization

–

+

Ca2+

NBCn1 or NBCn2

+

Na+

2 H+

Na+ 2 HCO3– HCO3– Na+

Depolarization

K+

PMCA

Increased [K+]o

+

NBCe1 Extracellular neuronal microenvironment

3 Na+ NBCe1 +

+

Depolarization K+

Decreased Na+

NSS +

[Na+]i

Decreased [Na+]i

–

2

2 HCO3

Alkalinization

B

Na-K pump

+

+

K+

Na+ NT–

ATP production

Glycolysis Astrocyte FIGURE 24. Role of NCBTs in excitable cells. Neuronal firing causes a depolarization induced alkalinization (DIA), via NBCe1, that anticipates and counters the dampening of neuronal excitability by Ca2⫹ pump-mediated H⫹ influx. The three electroneutral NCBTs also play critical roles in restoring neuronal pHi after a firing event (A). K⫹ released by firing neurons is absorbed by astrocytes causing a DIA, via NBCe1, that stimulates glycolytic ATP production (B), anticipating the increased energetic demand of the astrocyte for secondary active neurotransmitter (NT) uptake via neurotransmitter/sodium symporters (NSS).

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II) Central nervous system. A) Enhancement of neuronal excitability. Neuronal firing results in an intracellular acidification of neurons that tends to dampen neuronal excitability (reviewed in Refs. 76, 186, 187, and 898). The action of NCBTs in neurons, that as a population express at least NBCe1, NBCn1, NBCn2, and NDCBE, re-alkalinizes cells following firing events and thereby enhances the rate at which excitability recovers (FIGURE 24A). Indeed, mice lacking either of two other NCBTs, NDCBE and NBCn2, exhibit signs of reduced neuronal excitability.41 In hippocampal neurons under high-[K⫹]o conditions (a mimic of intense firing), the activity of NBCe1 is sufficiently strong that NBCe1 (and other factors) produce a depolarization-

MARK D. PARKER AND WALTER F. BORON induced alkalinization that overwhelms the natural tendency toward intracellular acidification (932).

III) Sensory organs. A) Transepithelial HCO3⫺ secretion across corneal endothelium. Working with a 1:2 stoichiometry (543) and importing HCO3⫺ from the stroma (FIGURE 19) into the cell, NBCe1 in the corneal endothelium is in a position to make a substantial contribution to the basolateral step of transepithelial HCO3⫺ secretion into the anterior chamber (i.e., aqueous humor). It is thought that this transcellular HCO3⫺ movement drives fluid reabsorption from the stroma into the anterior chamber, thereby maintaining appropriate corneal hydration and transparency (579, 922, 923, 1035).42 The molecular mechanisms underlying this process (shown in FIGURE 19) are reviewed in Reference 96. Briefly, cytosolic HCO3⫺ accumulates either as HCO3⫺ enters the cell directly across the basolateral membrane via NBCe1-B, or as HCO3⫺ forms from cytosolic CO2 (catalyzed by CA II) as Na-H exchangers extrude H⫹ across the basolateral membrane. Apical anion channels secrete HCO3⫺ into the anterior chamber. Thus the action of NBCe1-B helps provide cytosolic HCO3⫺ for secretion and also regulates pHi. Consistent with a contribution to fluid secretion by NBCe1, NBCe1 knockdown by siRNA reduces the transepithelial HCO3⫺ flux in cultured bovine corneal endo42 Although one model proposed by Wiederholt et al. places an electrogenic NCBT in the apical membrane, the authors state that this is an assumption since their methods did not allow them to determine the localization of NCBT in their cells (1035).

860

B) Potential to promote retinal attachment. In the retinal pigment epithelium, NBCe1-B is present in the apical membrane (94). NBCe1-mediated uptake of HCO3⫺ across the apical membrane would contribute to fluid absorption from the subretinal space to blood, presumably minimizing subretinal edema, as has been proposed for an apical electrogenic NCBT activity in bullfrogs (FIGURE 13A). Subretinal edema has not been described in Slc4a4-null mice nor in patients with NBCe1-associated pRTA, although it is possible that the presence of edema is masked by other ocular defects present in these individuals. IV) Peripheral nervous system. A) Neuronal excitability. In primary cultures of neurons from the rat trigeminal ganglion, application of anti-NBCe1-B/C siRNA results in a ⬃50% reduction of NBCe1-B/C protein abundance and, following an NH4⫹ prepulse, causes a near total elimination of HCO3⫺-dependent acid-extrusion in these cells (408). Thus NBCe1-B/C is likely to be the major NCBT in these cells. Furthermore, the frequency of action potential firing in response to current-injection in these cells is reduced by intracellular acidification and by DIDS treatment (408). Taken together, these data indicate that NBCe1-B/C mediates an uptake of HCO3⫺ that counters the dampening effect of intracellular acidification (408), FIGURE 24A pe and thereby plays an important role in maintaining excitability in these neurons. V) Circulatory system. A) Myocardial contractility and excitability. An NCBT-mediated increase in pH i enhances the contractility of myocardium in mammals (160, 702, 972). Moreover, HCO3⫺-dependent alkalinization is enhanced by repeated depolarizations of cat papillary muscle (147), consistent with the involvement of electrogenic NBCs. The current carried by electrogenic NCBT activity modulates the shape of myocyte action potentials, shortening action-potential duration and contributing towards a hyperpolarized resting membrane potential (14, 1006). Rat cardiac myocytes transfected with an adenoviral vector designed to overexpress NBCe1 are reported to exhibit an altered beat rate compared with nontransfected cells, although the direction of the rate change is not reported, and overexpression of NBCe1 transcripts or protein is not demonstrated (649). The relative contributions of NBCe1 and NBCe2 to these processes are unresolved. The influence of the action of

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B) Dampening of neuronal excitability by astrocytes. As neurons fire action potentials, they release K⫹ into the extracellular microenvironment (FIGURE 24A). One effect of the resulting elevated [K⫹]o would be to enhance neuronal excitability. However, the action of the Na-K pump in astrocytes tends to remove this accumulated extracellular K⫹, thereby dampening neuronal firing. The Na-K pump also maintains a low astrocytic [Na⫹]i, thereby promoting Na⫹coupled neurotransmitter uptake. A second effect of the elevated [K⫹]o is the stimulation of glycolysis in astrocytes via a feed-forward mechanism that anticipates the energy requirements of the astrocyte Na-K pump (83). The link between elevated [K⫹]o and the stimulation of astrocyte glycolysis appears to be NBCe1. Under conditions of intense neuronal activity, substantial K⫹ release would cause an NBCe1-dependent DIA in astrocytes (825). The consequent pH-dependent increase in the activity of glycolytic enzymes stimulates ATP production (FIGURE 24B). The importance of NBCe1 for this pathway is demonstrated by 1) the blockade of the pathway by S0859 and 2) the absence of this pathway from astrocytes cultured from neonatal NBCe1null mice (825). As the action of NBCe1 produces the DIA in astrocytes, the concomitant decrease in extracellular pH would dampen neuronal excitability and decrease the ability of neuronal NCBTs to counter intracellular acidification, thereby preventing excessive firing (as in FIGURE 24A).

thelium (579). In one study, a partial in-vivo knockdown (i.e., 25%) of NBCe1 by shRNA in rabbit eyes was not sufficient to produce the expected corneal thickening without the additional pharmaceutical inhibition of carbonic anhydrases (593). Given the mild knockdown, perhaps this result is not surprising. However, even in NBCe1-null mice, the effect of NBCe1 deficiency on HCO3⫺ secretion by colonic mucosa is detectable only following CA inhibition, even under secretagogue stimulated conditions.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

NBCe1 and other acid-base transporters on cardiac myocyte function is reviewed in Reference 996. VI) Musculoskeletal system. A) Myocyte contractility. By contributing towards pHi regulation in myocytes, NBCe1 likely contributes towards maintenance of contractility and excitability, as it does in cardiac myocytes (see above).

NBCe1 dysfunction is associated with enamel defects. Two alternative models have been proposed to explain how NBCe1 contributes to enamel formation. Both models posit that NBCe1 supports AE2-mediated HCO3⫺ secretion into the enamel surface compartment to buffer the H⫹ formed by apatite formation (706). However, the models differ in the location of NBCe1 and AE2, as well as in the consideration of how the enamel organ cells interact. In the first model (not shown), ameloblasts express NBCe1 in their basolateral membrane and AE2 (unusually for an Slc4) in their apical membrane. The concerted action of NBCe1-mediated HCO3⫺ influx and AE2-mediated HCO3⫺ efflux are proposed to form a pathway that secretes HCO3⫺ into the enamel-surface compartment (706). The second model (shown in FIGURE 20) is based on an alternative distribution of NBCe1 and AE2, and considers that the papillary cells and ameloblasts form a syncytium (456). In this model, NBCe1 is present in the membranes of papillary cells that abut the basal surface of ameloblasts, and AE2 is located in the basolateral membranes of ameloblasts (126, 456, 617). During the morphological switch of ruffle-ended ameloblasts to smooth-ended ameloblasts (associated with neutralization of the enamel-surface compartment acidity) a rearrangement of tight junctions exposes the lateral, but not the basal, surface of these cells to the enamelsurface compartment. Thus the action of papillary cell NBCe1, translated via gap junctions to the cytoplasm of the ameloblasts, is still in a position to support ameloblast HCO3⫺ secretion into the enamel fluid via AE2 (456). In an update to the second model, pendrin (Slc26a4) has been immunolocalized to the apical membranes of ameloblasts, providing an apical exit route for HCO3⫺ (125). However, unlike mice with AE2 or NBCe1 dysfunction, mice with a pendrin deficiency do not exhibit obvious defects in enamel deposition (125). HCO3⫺

secretion in the parotid B) Transepithelial fluid and salivary gland. Acinar cells in the parotid glands secrete an

In the duct cells, where basolateral NBCn1 is abundant ductal NBCe1 could contribute to the support of transepithelial secretion of HCO3⫺, via a mechanism similar to that described for NBCe1 in corneal endothelia (FIGURE 19). The role of basolateral NCBT activity in HCO3⫺ and fluid secretion in salivary glands is reviewed in Reference 555. (FIGURE 21B),

C) Protection of gastric mucosa from acid attack. The presence of NBCe1 transcripts in mammalian stomach preparations suggests that NBCe1 could, as has been proposed for an electrogenic NCBT in amphibian gastric mucosa (FIG⫺ URE 13B), support HCO3 secretion into the mucus layer that covers the stomach lining, thereby protecting gastric epithelia from acid attack. VIII) Lower digestive system. A) Transepithelial HCO3⫺ secretion across pancreatic duct cells. Working with a 1:2 stoichiometry and importing HCO3⫺ into a cell, NBCe1 in pancreatic duct cells can make a substantial contribution to the basolateral step of transepithelial HCO3⫺ secretion, and thus fluid secretion (422, 883, 1096). The contribution of NBCe1 towards the formation of pancreatic juice by acinar and duct cells is likely identical to that shown in FIGURE 21 for salivary glands. Cytosolic HCO3⫺ enters the cell either through NBCe1-B or is generated de novo by CA II action upon CO2. Apical Slc2643 proteins secrete HCO3⫺ into the duct lumen in exchange for Cl⫺. When fully stimulated by secretagogues (e.g., secretin), the luminal fluid in humans can be near-isotonic NaHCO3. The alkaline duct fluid keeps the pancreatic digestive enzymes in an inactive state and flushes them from the ducts, both of which protect from pancreatitis. In addition, pancreatic juice neutralizes acidic gastric chyme. NBCe1 has also been suggested to contribute towards the endocrine function of the pancreatic islets. The role of basolateral NCBT activity in ductal fluid and HCO3⫺ secretion is reviewed in Ref. 555. B) Transepithelial HCO3⫺ secretion across intestinal enterocytes. HCO3⫺ secretion across duodenal enterocytes plays a major role in protecting mucosa from acid attack (17). Con43 Anion secretion across the apical membrane of pancreatic duct epithelia is likely mediated by the concerted actions of CFTR, Ca2⫹activated chloride channels, and Slc26a6 (aka PAT1) with Slc26a3 (aka DRA1) playing a supporting role (333, 421, 628, 909, 1028).

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VII) Upper digestive system. A) Role in enamel deposition. The role of enamel organ epithelia in the formation of enamel in the enamel-surface compartment is still largely unknown. The apical membranes of ameloblasts face the enamel-surface compartment and, when mature, form alternating zones of ruffle-ended cells (facing enamel fluid that is acidic) and smooth-ended cells (facing enamel fluid that has a neutral pH).

isotonic fluid. The composition of the fluid is modified by duct cells that, among other functions, secrete proteins and HCO3⫺ to produce the HCO3⫺-rich saliva that acts to optimize amylase activity and to buffer gastric juices. Working with a 1:2 stoichiometry and importing HCO3⫺ into a cell, basolateral NBCe1 in parotid acinar cells is in a position to regulate pHi. Moreover, in concert with AE2, which would recycle HCO3⫺ back into the interstitium, the NBCe1 could make a contribution to the basolateral step of transepithelial NaCl and fluid secretion (Ref. 710, as shown in FIGURE 21A).

MARK D. PARKER AND WALTER F. BORON

C) Potential role in drug resistance of colon cancer cells. siRNA suppression of NBCe1 expression in a human colon carcinoma cell line increases sensitivity of the cells to the anti-cancer agent methotrexate (642), a phenomenon that the authors of the study hypothesize to be due to pH dependence of methotrexate uptake transporters. D) Potential role in transepithelial HCO3⫺ secretion across cholangiocytes in the liver. In cholangiocytes, immunolocalization studies seem to indicate that AE2 has an unusual apical disposition (46, 631, 855, 902, 971). Moreover, it has been proposed that this apical AE2 mediates HCO3⫺ secretion into the bile duct lumen (46, 631, 902), thereby protecting the liver from bile acid attack (390). In the cholangiocytes of mice that are unable to express the a and b variants of AE2, NBCe1 transcript and protein abundance are increased compared with control cells from wild-type mice, as is an electrogenic NBC activity (987). As cholangiocytes of AE2a,b-null mice are able to compensate for their HCO3⫺ secretion deficit via a Na⫹-dependent mechanism, it is suggested that NBCe1, again targeted to the apical rather than the basolateral membrane, might be able to compensate for a HCO3⫺ secretion defect by operating with a 1:3 stoichiometry (987).44 Neither the apical presence of NBCe1 protein in mouse cholangiocytes nor the stoichiometry of the transport process in these cells has yet been demonstrated, although NBCe2 immunoreactivity has been detected in the apical membrane of rat cholangiocytes (8).

44 Although rat cholangiocytes express NBCe2 in their apical membranes (8), mouse cholangiocytes are reported to lack NBCe2 (987).

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IX) Lymphatic and immune systems. The lymphatic and immune systems are not major sites of NBCe1 expression. We are unaware of any reports that assign a physiological role to NBCe1 in these systems. X) Endocrine system. A) Possible role in HCO3⫺ exit from pancreatic islet cells. Insulin-producing cells generate a substantial amount of CO2 that is linked to the production of insulin. One group suggests that the CO2 generated from nutrient insulin secretagogues (e.g., glucose) first is converted to HCO3⫺ for exit across the plasma membrane (863). NBCe1 is expressed in both pancreatic islet cells and a related tumor cell line (135, 901). Although both NBCe1-A and NBCe1-B are present in islets, NBCe1-B predominates in the insulin-producing ␤ cells. Treatment with tenidap (an inhibitor of NBCe1) reduces glucose metabolism, reduces glucose-stimulated insulin secretion, and also lowers pHi. The last observation is consistent with the hypothesis that NBCe1-B normally functions as an acid extruder (i.e., mediates HCO3⫺ uptake) in these cells. However, tenidap also increased 22Na uptake and hyperpolarized the cells, which would be consistent with the opposite hypothesis: that NBCe1-B normally operates as an acid loader (i.e., mediating HCO3⫺ efflux). It seems clear that NBCe1 is important for maintaining insulin secretion from pancreatic tissue, by promoting fluid secretion. However, the tenidap (which was developed by Pfizer as a nonsteroidal anti-inflammatory drug) probably has complex actions in these cells, witness the effects on 22Na fluxes and Vm. In any case, we would not expect the CO2 generated from the metabolism of nutrient secretagogues to exit the cell via NBCe1, which is presumably mediating the net uptake of HCO3⫺. Even in the renal PT, which generates large amounts of CO2 and in which NBCe1-A mediates HCO3⫺ efflux, the most straightforward mechanism for the disposal of metabolically generated CO2 is the same as for other cells in the body: CO2 in the steady state moves passively across the cell membrane, perhaps via gas channels, and diffuses into systemic capillaries for disposal in the exhaled air. For a discussion of the contribution of NBCe1 to the digestive role of the pancreas, see above. XI) Urinary system. A) HCO3⫺ reabsorption across proximal tubule epithelia. As illustrated in FIGURE 23, NBCe1-A plays a central role in the transepithelial secretion of H⫹ by the renal PT. H⫹ extruded across the apical membrane has three fates, titrating: 1) HCO3⫺ (filtered from blood in the glomerulus) to CO2 ⫹ H2O, 2) NH3 to NH4⫹, and 3) HPO42– (and weak bases other than HCO3⫺ and NH3) to H2PO4– (and the conjugate weak acids of the other weak bases), the so-called titratable acidity. In the case of HCO3⫺ reabsorption, the newly formed CO2 and H2O enter the PT and form HCO3⫺. In the case of NH4⫹ excretion and formation of titratable acidity, the intracellular HCO3⫺ forms from CO2 that originates either from PT oxidative metabolism or from the blood. The common denominator is that

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sistent with a role for NBCe1-B in HCO3⫺ secretion throughout the gut (via a mechanism such as that shown for a duodenal villar enterocyte in FIGURE 22), the secretagogues carbachol and forskolin increase the basolateral abundance of NBCe1 protein in rat proximal jejunum enterocytes (430) and in murine colonic crypts (1087). Furthermore, both DIDS and siRNA knockdown of NBCe1 inhibit parathyroid-hormone–stimulated short-circuit currents, a measure of transepithelial anion secretion, across monolayers of a human intestinal epithelial-like cell line (172). On the other hand, compared with tissues from wildtype mice, proximal colons from NBCe1-null mice exhibit a reduced cAMP-stimulated HCO3⫺ secretion only under conditions in which blockade of CA II severely curtails the generation of intracellular HCO3⫺ from CO2 (313). At face value, these data are inconsistent with the idea that NBCe1 plays a major role in HCO3⫺ secretion under physiological conditions. On the other hand, it is possible that the NBCe1-null mice may have upregulated the CA-dependent pathway by enhancing basolateral H⫹ extrusion via NHEs or NBCn1. At least in the duodena of mice, NBCn1 makes a more substantial contribution to HCO3⫺ secretion than NBCe1.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

NBCe1-A exports the HCO3⫺ across the basolateral membrane. The role of an electrogenic NCBT as the basolateral step in the pathway that reabsorbs HCO3⫺ from the PT lumen was first demonstrated in salamanders in 1983 (103). Demonstration of the equivalent activity in mammals, namely, rabbits and rats, followed in an array of papers published between 1985 and 1987 (28, 80, 81, 331, 514, 832, 896, 1085). The subsequent cloning and characterization (809) as well as the immunolocalization of NBCe1 to the basolateral membranes of PT epithelia (632, 844) demonstrated that NBCe1 is indeed the transporter responsible for this activity.

45 Because NBCe1 knockout mice have not survived to a breeding age (313), it is not known whether NBCe1 is necessary for fertility.

B) Transepithelial HCO3⫺ secretion across uterine epithelia. Basolateral NBCe1 is in a position to support HCO3⫺ secretion across endometrium epithelia (1027) that secrete a HCO3⫺-rich uterine fluid, which is important for sperm capacitation and egg fertilization (e.g., see Ref. 554). H) CAUSES OF NBCe1 UPREGULATION.

In this section we consider disturbances that result in upregulation of NBCe1 at the level of transcript abundance, protein abundance, translocation to the plasma membrane, or transporter activity. Note that an increase in any one of these factors need not necessarily correlate with an increase in the others. The plasma-membrane abundance, as well as per-molecule activity, of NBCe1-B/C can be increased by activation of the soluble binding partner IRBIT. However, the physiological cues that activate IRBIT have not been described. In the following discussion, we have omitted cellular studies that report only indirect evidence of NBCe1 upregulation (e.g., upregulation of HCO3⫺ reabsorption) because such observations might at least in part be explained by effects on other proteins. We have arranged the reports in the order of the organ in which each observation was made and then in order of disturbances that are shown to increase NBCe1 transcript abundance, increase NBCe1 protein abundance, increase NBCe1 abundance in the plasma membrane, and stimulate NBCe1 activity. I) Central nervous system. A) Increased transcript abundance following cerebral arterial occlusion. In rats subjected to permanent cerebral-artery occlusion, the abundance of NBCe1 protein in the ischemic penumbra is more than twice as great as in sham-operated controls (458). It is reasonable to suggest that this upregulation of NBCe1 leads to an increase in [Na⫹]i that could contribute to edema as well as other secondary brain injuries. Thus NBCe1 inhibitors have the potential to limit such ischemic damage (458). B) Increased protein abundance following seizure induction. Seizure-sensitive and seizure-resistant gerbils exhibit similar expression patterns for NHE1 and NBCe1 immunoreactivity. However, 30 min and 180 min after the induction of seizures in the SS gerbils, the expression of both proteins increased markedly in the hippocampal CA1–3 regions and granule layer of the dentate gyrus (467). Also, NBCe1 protein levels are elevated in the hippocampi of gerbils 4 h after administration of the GABAB receptor agonist baclofen but not after administration of the GABAA receptor agonist muscimol (466).

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XII) Reproductive system. A) Possible role in HCO3⫺ reabsorption and/or secretion in the epididymis. The lumen of the epididymis is a site of Na⫹ reabsorption and H⫹ secretion (572), with low luminal pH being a requirement for storage of viable sperm. NBCe1-B is present in the cells of the epididymis, and cultured epididymal cells exhibit a DIDS-sensitive, Na⫹- and HCO3⫺-dependent pHi recovery from an acid load, leading several groups to suggest that NBCe1 might be involved in H⫹ secretion/HCO3⫺ reabsorption by these cells (164, 445, 729, 1119). Mice deficient in the estrogen receptor ESR␣ (or ESR1) are defective in their ability to acidify the epididymal lumen. In the initial segment of the epididymis, these mice exhibit a ⬃50% reduction in protein abundance of apical NHE3 and CA XIV, as well as basolateral NBCe1 (453). At present there is no direct evidence that NBCe1 plays a substantial role in epididymal HCO3⫺ reabsorption in these cells. Relevant issues include: 1) the direction of NBCe1-mediated transport can vary in a tissue-specific manner (346) and physiological data to support an outwardly directed basolateral NCBT activity in these cells is presently lacking. 2) In order for a basolateral NBCe1-B to contribute to HCO3⫺ reabsorption, it would presumably have to operate with a 1:3 stoichiometry, rather than the 1:2 stoichiometry that it has in pancreatic ducts (344). On the other hand, one report suggests that the stoichiometry of NBCe1-B might depend on the celltype in which it is expressed (346). 3) NBCe1 immunoreactivity in the epididymis is not restricted to the acid-secreting narrow or clear cells (729). 4) AE2, a related acid-loading transporter, is also expressed in the basolateral membranes of epididymal epithelia (446). Mice that are unable to express the a, b1, and b2 variants of AE2 are infertile (638). Thus NBCe1-B is unable to compensate sufficiently in these knockouts.45 On the other hand, luminal H⫹ secretion from epididymal epithelia is stilbene-sensitive and independent of Cl⫺ (120, 164, 1119). We conclude that the physiological role of NBCe1-B in epididymal H⫹ secretion/HCO3⫺ reabsorption remains open. One possibility is that NBCe1-B plays

a role in pHi regulation in epididymal epithelia. Another possibility is that NBCe1-B supports regulated HCO3⫺ secretion in epididymal epithelia (152, 164), which could activate sperm mobility prior to ejaculation (697, 937).

MARK D. PARKER AND WALTER F. BORON

II) Circulatory system. A) Increased transcript abundance and activity in heart following abdominal aortal constriction. NBCe1 (and NBCn1) transcript abundance increases in a rat model of ventricular hypertrophy (1071), generated by constriction of the abdominal aorta, and is accompanied by an increase in HCO3⫺-dependent acid extrusion in myocytes isolated from the hypertrophic ventricles. The authors suggest that NBCe1 contributes to an increased Na⫹ load in hypertrophic myocytes, promoting arrhythmia and reperfusion injury via activation of the Na-Ca exchanger (1071). The action of NBCe1 also could contribute towards the severity of the hypertrophy in myocytes, as described for NHE1 (676, 1061). Indeed, overexpression of cardiac NBCe1 may exacerbate reperfusion injury by contributing to ischemic [Na⫹]i overload (956). B) Increased transcript and protein abundance in heart following terminal heart failure. Cardiac NBCe1 transcript and protein levels are both elevated in preparations from individuals that suffered terminal heart failure, although whether this is a cause or consequence of heart failure has yet to be established (481).46 C) Increased protein abundance in heart by chronic hypercapnia. In neonatal, but not adult mice, chronic (2 wk) exposure to 12% CO2 causes NBCe1 protein abundance to increase by ⬃40% in heart, reflecting a general pattern of

46 The authors of another study report a significant difference in NBCe1 mRNA expression levels between normal and failing hearts based on microarray data, but do not report the direction of the change (649). Transfecting cultured rat cardiomyocytes with an adenoviral vector that is designed to overexpress NBCe1 “modified the beating rate” and “lowered the viability,” although no confirmation of NBCe1 overexpression or primary data were provided (649). Mice genetically modified with an NBCe1 transgene (to mimic NBCe1 overexpression) exhibited no cardiac detectable or bloodpressure phenotype, although neither the identity of the splice variant nor confirmation of NBCe1 overexpression was provided (649).

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increased abundance of acid extruders (e.g., NBCn1 and NHE1), which may help to counter the acidifying effects of hypercapnia (463). Also in the kidney, hypercapnia increases NBCe1 protein abundance. D) Potential stimulation of NBCe1 in response to ethanolinduced acidosis. The application of 30 –1,000 mM ethanol to human atrial cardiac myocytes causes a graded fall in pHi and a modest stimulation of an unidentified HCO3⫺-dependent acid extruder (979), likely NBCe1. Note that even 30 mM ethanol is about twice the legal limit for alcohol intoxication in many jurisdictions. Moreover, the study did not take into consideration either the osmolality or reflection coefficient of ethanol. E) Increased activity in cardiac myocytes in response to acidosis and/or angiotensin II. In cardiac myocytes (1072), acute intracellular acidosis stimulates an unidentified electrogenic NCBT, likely NBCe1, that contributes to pHi recovery. In infarcted rat hearts, acidosis increases the abundance of NBCe1 transcripts and protein in the left ventricular free wall via a pathway that involves a local renin-angiotensin system, including angiotensin converting enzyme, angiotensin II (ANG II), and stimulation of AT2 receptors (831). Stimulation of NCBT activity by 10⫺7 M ANG II via an AT2-dependent pathway has also been demonstrated in neonatal rat cardiac myocytes in which the stimulation can be mimicked by application of arachadonic acid (503). Other studies report that the stimulatory effect of 10⫺7 M ANG II upon NCBT activity in rat (313)and cat cardiac myocytes is mediated by the AT1 receptor (58, 224), similar to the stimulation of NBCe1 functional expression in the proximal tubule. However, in cat cardiac myocytes, the phenomenon stimulated by ANG II is reported to represent stimulation of NBCn1 and inhibition of NBCe1. We note in summary that the study of Sandmann and coworkers (831), in which ANG II stimulates NBCe1, is corroborated by molecular evidence of the increased NBCe1 abundance. On the other hand, the presence of NBCn1 in cardiac myocytes is not well demonstrated. Without invoking species differences, these studies are not readily reconciled. III) Musculoskeletal system. A) Increased protein abundance in skeletal muscle following training. NBCe1 protein abundance is doubled in the soleus muscle (a predominantly oxidative organ), but not the extensor digitorum longus muscle (a predominantly glycolytic organ), of rats after 5 wk of interval training on a treadmill (964). B) Increased protein abundance in skeletal muscle at high altitude. Human subjects that live at high altitude, or those who normally live at low altitude but move to high altitude

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An elevation of NBCe1 protein abundance also occurs in gerbils treated with the GABA degradation inhibitor vigabatrin (466). Two critical issues not addressed in the aforementioned studies are the identity of the upregulated NBCe1 splice variant (i.e., NBCe1-B versus -C) and the identity of the cells in which it was upregulated. For example, in rat hippocampus, NBCe1-C is abundant in the astrocytes that surround the neuronal cell bodies in the pyramidal cell layer (624). If the seizure activity leads to an increase in NHE1 and NBCe1-C activity in astrocytes, that would lower extracellular pH and reduce neuronal excitability (reviewed in Refs. 186, 187, and 898). However, if the seizure activity leads to an increase in NHE1 and NBCe1-B in neurons, that would tend to increase neuronal excitability, which would be a maladaptive consequence of an attempt to protect neurons from acidosis.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

for 8 wk, have double the abundance of NBCe147 protein in their skeletal muscle compared with individuals who live at sea level (457). The authors of the study suggest that this upregulation of NBCe1 as an acid extruder may reflect a mechanism that compensates for lower-than-normal arterial HCO3⫺ (at rest), itself a compensation for the respiratory alkalosis produced by hyperventilation, measured in these individuals at high altitudes (457). If it turns out that CO32⫺ is the substrate of NBCe1 (p. 56), then a critical question is whether the combination of the uncompensated alkalosis (i.e., high pHo) and compensatory low [HCO3⫺]o results in a depressed [CO32⫺]o.

B) Increased transcript abundance during dark periods. Investigators studying the circadian rhythm of transcript abundance in mouse molars determined that NBCe1 transcript abundance is nearly doubled during dark periods compared with light periods (537). The authors suggest that this observation correlates with periods of enhanced enamel deposition by ameloblasts (537). V) Lower digestive system. A) Increased protein abundance in the plasma membrane of colonic mucosa by secretagogues. The application of forskolin, which raises [cAMP]i, stimulates colonic HCO3⫺ secretion in vivo, without increasing NBCe1 transcript abundance (55), by enhancing the accumulation of NBCe1-B protein in the plasma membrane (1087). In contrast, forskolin inhibits heterologously expressed NBCe1-B activity in a renal cell line (54). VI) Urinary system. A) Increased transcript abundance by chronic dexamethasone treatment. Glucocorticoid excess causes a metabolic alkalosis associated with enhanced renal

47 In this report, the authors state that the antibody used in this study (no. 3212; Chemicon) does not discriminate between NBCe1, NBCe2, or NBCn1. The antibody was raised against an 54-amino acid epitope in the soluble Nt domain of rat NBCe1 and appears, as evidenced by lack of immunoreactivity with the mTAL in rat kidney section (844), to be at least unreactive towards NBCn1. A later paper by the same group demonstrated that this antibody and an NBCe2-specific antibody recognize proteins of different molecular weights in rat muscle preparations, indicating that this antibody is likely to be specific for NBCe1.

B) Increased transcript and protein abundance following birth. In mice, NBCe1 transcript and protein abundance increases in the PT following birth (in this study, day 3 to day 18), coordinated with the upregulation of other renal ion transporters, such as NHE3, and concomitant with a drop in urinary pH over the same time period (97). C) Increased transcript and protein abundance following renal transplant rejection. NBCe1 transcripts and protein levels are increased in PTs of transplanted rat kidneys following acute rejection (999). The significance of these findings is presently unclear. D) Increased transcript abundance in Aadc-null mice. Mice with a PT-specific deletion of aromatic amino acid decarboxylase (AADC) exhibit elevated NBCe1 mRNA abundance (1094). Because AADC catalyzes the final step in dopamine synthesis, and because dopamine reduces Na/ HCO3 cotransport activity, these observations suggest that it is intrarenal dopamine-signaling pathways that normally limit NBCe1 abundance. E) Increased transcript abundance and stimulation of activity in K⫹-deprived rats. Increased NBCe1 transcript abundance and elevated NCBT activity has been described in the PT and medullary thick ascending limb (mTAL) of K⫹deprived (KD) rats. Upregulation of HCO3⫺ reabsorption by these tubule segments has been implicated in the pathogenesis of whole-body alkalosis in KD animals (38, 894). For the mTAL, this hypothesis requires that the NBCe1, the splice variant of which is unknown, be basolateral and operate in the HCO3⫺-outward direction. The medulla is not usually associated with NBCe1 expression (38); mTAL epithelia normally only express the relatively DIDS-insensitive NBCn1. In the renal medulla of KD rats, NBCe1 transcript abundance is increased approximately fourfold, and an unusual DIDS-sensitive NCBT activity is detected in mTAL epithelia (38). A whole-kidney intracellular acidosis, measured by 31P-NMR, has also been noted in KD rats (10). To the extent that this “renal” pHi decrease reflects a fall in the pHi of PT cells, it would be consistent with an increase in basolateral NBCe1-A activity, which would in turn tend to alkalinize the blood. F) Increased protein abundance by chronic norepinephrine treatment. Consistent with a potentially causative role in norepinephrine-promoted Na⫹ retention, NBCe1 protein abundance is doubled in the renal cortex of norepinephrineinfused rats, as is the abundance of two other Na⫹ trans-

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IV) Upper digestive system. A) Increased transcript abundance in ameloblast-like cells maintained at acidic pH. LS8 cells are derived from the enamel organ of embryonic mice. In LS8 cells maintained for 24 h in acidic medium, NBCe1 transcripts are more abundant than in LS8 cells maintained in an alkaline medium (706, 891). This phenomenon is controlled by a pH-responsive enhancer region in the NBCe1-B/C promoter. If the increased abundance of NBCe1-B transcripts in LS8 cells translates to an increase in NBCe1 acid-extruding activity (see FIGURE 20), enamelorgan cells exposed to acid should be adapted to 1) defend pHi from acidosis and 2) secrete HCO3⫺ into acidic enamel fluid.

HCO3⫺ reabsorption. Consistent with this phenomenon, a 4-day period of dexamethasone treatment results in a doubling of NBCe1 transcript abundance in the renal cortex of rats (21).

MARK D. PARKER AND WALTER F. BORON porters, namely, the Na/K/Cl cotransporter 2 (NKCC2) and NHE3 (899). G) Increased protein abundance in hypovolemic rats. The observed increase in NBCe1 and NHE3 protein abundance in the PT epithelia of hypovolemic (i.e., volume depleted) rats might contribute to the increased urine acidity in these animals (620). These changes would promote fluid reabsorption and thus be a reasonable adaptation to hypovolemia.

According to the first report, NBCe1 protein abundance is doubled in the renal cortex of SHR compared with control rats (900). With the assumption that this change corresponds to an increase in functional NBCe1 activity, the result would be increased Na⫹ reabsorption, which would be expected to contribute toward a hypertensive phenotype. According to the second report, a study of immortalized PT epithelia from SHRs, NBCe1 transcript abundance and NCBT activity are reduced (731).48 These results may seem counterintuitive because downregulation of NBCe1 would tend to reduce, not increase, Na⫹ reabsorption. However, reduction of NBCe1 in these immortalized cells may reflect an adaptation to hypertension that occurred in the donor SHR rat. Possible explanations for the apparent discrepancy between the two studies include 1) differences between rat tissue and immortalized cell lines, 2) variability in the genetic basis of hypertension (e.g., in one case NBCe1 contributes to hypertension whereas in the other it opposes it) between populations of SHR rats (672), and 3) an increase in NBCe1 protein abundance may not result in an increase in NCBT activity. I) Increased protein abundance in the kidney during chronic hypercapnia. In adult rats, chronic (10 day) exposure to 8% CO2, 13% O2 (i.e., hypoxic hypercapnia) results in a near 48 Interestingly, the characteristics the NCBT activity in immortalized SHR cells differ from the NCBT activity in control cells in two ways (731). In SHR cells, NCBT activity is 1) more sensitive to stimulation by acidosis and 2) poorly DIDS-sensitive (50% blockade by 1 mM DIDS). These features are reminiscent of NBCn1 which is strongly upregulated by acidosis and poorly sensitive to DIDS, leaving open the possibility that NBCn1 is expressed in these immortalized SHR cells.

866

J) Increased plasma membrane abundance in the kidney in response to ANG II. In the proximal tubule, low doses of ANG II stimulate reabsorption of HCO3⫺ (JHCO3) and Na⫹ (e.g., see Refs. 367, 394, and 1106). A 15-min application of 10⫺10 M ANG II to a polarized monolayer of immortalized renal epithelial cells from opossums (OK cells) increases the basolateral plasma membrane abundance of NBCe1 (802). The enhancement of NBCe1 functional expression by ANG II in these cells is blocked by antagonists of the AT1 receptor, blockers of Src family tyrosine kinases, and blockers of the mitogen-associated protein kinase (MAPK) signaling pathway (802).49 Furthermore, the ANG II–induced increase in JHCO3 is absent in AT1A receptor-null mice (394, 1100). As shown in studies of perfused tubules, and as modeled in Xenopus oocytes, the effects of ANG II are biphasic; low concentrations (10⫺10 and 10⫺11 M) of ANG II are stimulatory to NBCe1 functional expression, whereas higher concentrations are inhibitory (367, 394, 738, 739). In isolated perfused rabbit proximal tubules, acute isolated increases in basolateral [CO2] or isolated decreases in basolateral [HCO3⫺] cause an increase in JHCO3 (1108). This response requires the secretion of local ANG II into the tubule lumen (1104) and is blocked by inhibition or knockout of luminal AT1 receptors (1107). Note that agonism of the M1 muscarinic receptor, another G protein-coupled receptor, is associated with an increase in Na⫹ and HCO3⫺ reabsorption by the PT (823). The action of the non-receptor tyrosine kinase Pyk2 appears to be a common factor in the costimulation of NBCe1 and NHE3 activity by GPCR agonists and by acidosis (277, 582). ANG II also enhances functional expression of NBCe1 in the heart via an AT2-dependent pathway that appears to share commonality with the pathway that upregulates NBCe1 in response to acidosis (see p. 866).

49 Src and MAPK phosphorylation are also implicated in a PPAR␥associated pathway that stimulates NBCe1 functional expression in response to thiazolidinedione treatment.

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H) Increased protein abundance in some spontaneously hypertensive rats. Dopamine normally reduces Na/HCO3 cotransport activity in the proximal tubules of rabbits and rats (524). In a particular strain of spontaneously hypertensive rats (SHRs), an undetermined defect in the DA1 dopamine receptor leaves presumed NBCe1 activity unresponsive to downregulation by dopamine (524), thereby limiting the ability of the PT to reduce Na⫹ reabsorption (405). Two other reports that directly address perturbation of NBCe1 in SHRs appear to differ in their findings.

doubling of NBCe1 protein abundance in the PT (226). This observation is consistent with the findings of an earlier study of cultured rat proximal tubules cells in which stimulation of Na/HCO3 cotransport activity by respiratory acidosis was prevented by treatment with inhibitors of protein synthesis (824). In neonatal, but not adult mice, chronic (2 wk) exposure to 12% CO2 causes NBCe1 protein abundance to increase by ⬃20% in kidney (463). These responses reflect a general pattern of increased abundance of acid extruders (e.g., NBCn1 and NHE1), which may help to counter the acidifying effects of hypercapnia (463). Hypercapnia also increases NBCe1 protein abundance in the heart (p. 877).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

K) Stimulation of activity in response to acidosis. In the renal cortex of rats, NBCe1-A transcript (140) and protein (36, 484, 530) levels are unperturbed by NH4Cl-induced acidosis. However, consistent with a model in which existing NBCe1 protein is activated, NBCe1 activity is increased in acidotic rats (757) and rabbits (15), isolated basolateral membrane vesicles prepared from suspensions of rabbit PTs subjected to metabolic acidosis (895), and immortalized rat PT epithelia treated with NH4Cl (731). Transcript abundance of an unidentified NBCe1 variant in an inner medullary collecting duct (IMCD) cell line is decreased by acid stress (1029); however, the IMCD is not a site of substantial NBCe1 expression. Acidosis also stimulates NBCe1 activity in the heart.

XIII) Stimulation of functional expression in PT by thiazolidinediones. Drugs such as pioglitazone (PGZ) and rosiglitazone (RGZ), agonists of peroxisome proliferator-activated receptor gamma (PPAR␥), are used to increase insulin sensitivity in patients with type II diabetes. Thiazolidinedione (TZD) use is associated with an expansion of plasma volume that is hypothesized to be due to increased renal solute reabsorption (272). Endo and co-workers (272) report that PGZ and RGZ stimulate basolateral HCO3⫺ transport in rabbit PTs via a PPAR␥-dependent pathway that also increases the phosphorylation of Src family kinases and MAPK. Although the mechanism of increased basolateral HCO3⫺ transport in response to TZD treatment is untested in this instance, other studies link the activation of Src and MAPK pathways with an increase in the plasma membrane abundance of NBCe1 in the PT (see p. 868). I) CAUSES OF NBCe1 DOWNREGULATION. In this section we consider disturbances that result in downregulation of NBCe1 either at the level of transcript abundance, protein abun-

50 Conversely, NaCl feeding, a maneuver that would tend to lower the glomerular filtration rate, results in a reduction of NBCe1 protein abundance.

I) Central nervous system. A) Reduced protein abundance in brain in response to intermittent hypoxia. Chronic intermittent hypoxia (CIH), a model for sleep apnea, appeared to decrease NBCe1 protein abundance in the cerebellum as assessed with an antibody that should not discriminate among NBCe1 variants (261). On the other hand, antibodies specific for NBCe1-A/B and NBCe1-C did not reveal statistically significant changes in the cerebellum. II) Circulatory system. A) Potentially reduced activity in cardiac myocytes in response to angiotensin II. Although studies from two groups of investigators are consistent with upregulation of NBCe1 in rat cardiac myocytes by ANG II (via an AT2-dependent pathway), studies by a third group are consistent with downregulation of an NBCe1-like activity by the same concentration of ANG II in cat cardiac myocytes (via an AT1-dependent pathway). These observations are not readily reconciled, but could be explained by species differences. III) Upper digestive system. A) Reduced transcript abundance in ameloblast-like cells maintained at alkaline pH. NBCe1 transcripts are less abundant in LS8 cells (derived from the enamel organ of embryonic mice) that are maintained for 24 h in alkaline medium (891). This adaptation is the counterpart to the upregulation of NBCe1 in response to decreased extracellular pH, which is proposed to support ameloblast function. IV) Lower digestive system. A) Downregulation in jejunum by chronic gamma-irradiation. Diarrhea such as that associated with radiation exposure follows a decrease in anion (and thus fluid) absorption and/or an increase in anion/fluid secretion. In the case of secretagogue-induced diarrhea, anion secretions are rich in Cl⫺ and HCO3⫺, supported by upregulation in the gut of NKCC1 (211) and NBCe1. However, in gamma-irradiated mice, increased anion secretion is effected with a seemingly counterintuitive reduction in HCO3⫺ secretion (1093). In the jejuna of these mice, NBCe1-A/B immunoreactivity is lost, reducing support for HCO3⫺ secretion from jejunal enterocytes (1093). An accompanying increase in NKCC1 immunoreactivity in the

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L) Stimulation of activity in PT by chronic hyperfiltration. In rats, following removal of a kidney, the remaining kidney experiences a ⬃50% increase in glomerular filtration rate. In addition, the PTs from the remnant kidney adapt (2 wk after surgery) by doubling the functional expression of both apical Na-H exchange and basolateral Na/HCO3 cotransport (758).50 This adaptation does not appear to be an acute effect of acidosis inasmuch as blood pH was not different from that of control rats (758). Whether this upregulation of NBCe1 activity is accompanied by an increase in the abundance of NBCe1 transcripts and/or protein is not documented in this study. However, in rats in which one ureter is partially obstructed within 48 h of birth, NBCe1 protein abundance is doubled after 7 wk in both the obstructed kidney and unobstructed kidney (1025).

dance, translocation to the plasma membrane, or transporter activity. Note that a decrease in any one of these factors need not necessarily correlate with a decrease in the others. We have omitted cellular studies that report only indirect evidence of NBCe1 downregulation (e.g., inhibition of HCO3⫺ reabsorption) because such observations might at least in part be explained by effects on other proteins. We have arranged the reports in the order of the organ in which each observation was made and then in order of disturbances that are shown to reduce NBCe1 transcript abundance, reduce NBCe1 protein abundance, reduce translocation to the plasma membrane, and reduce NBCe1 activity.

MARK D. PARKER AND WALTER F. BORON jejuna of irradiated mice suggests that increased Cl⫺ uptake across the basolateral membrane may compensate for decreased anion secretion support from NBCe1 insufficiency, producing Cl⫺–rich secretions (1093).

C) Downregulation by microRNAs in colon carcinoma cells. In the human colon carcinoma cell line HT29 (642), the microRNA miR-224 is under-represented. The 3=-UTR of NBCe1 transcripts is a potential target of miR-224 (642). Consistent with this hypothesis, NBCe1 transcript abundance are unusually abundant in HT29 cells. V) Endocrine system. A) Decreased transcript abundance in thyroid cancer. Two studies report a greater than threefold reduction in NBCe1 transcript abundance in papillary thyroid carcinoma compared with normal thyroid tissue isolated from the same patients (309, 486). With the assumption that this downregulation correlates with a decrease in functional expression, and that in these cancer cells NBCe1 operates in the HCO3⫺-inward direction, these changes, which would tend to lower pHi, might be predicted to harm the cancer cell rather than encourage tumor proliferation (see below for a discussion of the reported downregulation of NBCn1 in breast cancer). VI) Urinary system. A) Decreased transcript and protein abundance and reduction of activity in kidneys of Na⫹loaded and alkalotic animals. In the PT epithelia of rats whose drinking water is spiked with NaHCO3 or NaCl (thereby producing a Na⫹ load that could make the animal hypervolemic), NBCe1 transcript (140) and protein (36, 620) abundance is reduced. These adaptations would tend to reduce Na⫹ reabsorption and thus tend to oppose the development of hypertension. The adaptations would also tend to reduce HCO3⫺ reabsorption, which in the case of NaCl feeding might render the animals less able to respond to an acute acid load. In the case of NaHCO3 feeding, the adaption would oppose whole body alkalosis (37). In rabbits, Cl⫺-depletion alkalosis (CDA), which presumably re-

868

B) Decreased transcript abundance and reduction of activity in PT of some spontaneously hypertensive rats. As discussed and contrasted above, although some spontaneously hypertensive rats exhibit increased NBCe1 protein abundance, authors of a separate study report reduced NBCe1 transcript abundance and reduced NBCe1 activity in another population of SHRs. Inasmuch as reduced NBCe1 activity would tend to counter hypertension by reducing Na⫹ reabsorption, these reductions are more likely to be a consequence than a cause of hypertension in these animals. C) Reduced protein abundance in response to hypoxia. An immunohistochemical study of mouse kidney slices appears to show, although the authors of that study do not specifically comment on this phenomenon, a reduced abundance of NBCe1 protein in the basolateral membranes of PT epithelia in slices that are briefly (10 s to 2 min) exposed to hypoxia prior to cryofixation (829). D) Decreased protein abundance in kidney following ureteral obstruction. Following a 24-h bilateral ureteral occlusion in rats, NBCe1 and NHE3 protein abundance is substantially decreased in the PTs (1024). This downregulation may partly explain the phenomenon of obstruction-induced renal tubular acidosis. In rats in which only one ureter is partially obstructed within 48 h of birth, NBCe1 protein abundance is doubled after 7 wk in both the obstructed kidney and unobstructed kidney (1025). On the other hand, after 14 wk, the obstructed kidney exhibits a ⬃40% decrease in NBCe1 protein abundance, whereas the abundance of NBCe1 in the unobstructed kidney appears to be close to normal (1025). E) Decreased protein abundance in kidney during gentamycin-induced nephropathy. Treatment of bacterial infections with gentamycin can cause PT dysfunction in humans (reviewed in Ref. 308). In rats treated with gentamycin for 7 days, renal NBCe1 protein abundance is decreased by half (57). The abundance of other renal Na⫹ transporters, namely, NHE3 and the Na-K pump, and the water/carbon dioxide channel AQP1 are also reduced under these conditions (57). J) CONSEQUENCES OF NBCe1 DYSFUNCTION. In this section we consider the pathologies that are associated with genetic ablation of, genetic alterations in, and dysfunction of NBCe1 in the brain, eye, heart, enamel organ, lower diges-

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B) Suggested reduction of activity in pancreas by elevated glucose levels. In isolated, perfused pancreatic ducts, high levels of luminal glucose lead to an accumulation of Na⫹ and membrane depolarization, via the action of the apical Na/glucose cotransporter SGLT1, in duct cells (307). The authors of the study suggest that elevated [Na⫹]i inhibits NBCe1-mediated influx of Na⫹ and HCO3⫺ at the basolateral membrane, thereby contributing to the decreased pancreatic HCO3⫺ secretion associated with diabetes. On the other hand, the authors suggest that the depolarization, via an inhibitory effect upon CFTR, could be a more important factor in reducing HCO3⫺ secretion. Depolarization would be expected to enhance NBCe1 activity. This model requires that elevated serum [glucose] results in an elevated luminal [glucose] via an undetermined transcellular glucose transport pathway.

sults in volume contraction, causes a fall of NBCe1 activity in basolateral renal cortical membrane preparations (15). On the other hand, in rats, a similar stress has no effect on NBCe1 transcript levels in the PT (140). If these two observations can be taken together, they are consistent with the hypotheses that 1) the regulation of NBCe1 activity by alkalosis is purely posttranslational or 2) the response to CDA is species specific.

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

tive system, and kidney. The primary syndrome associated with NBCe1 dysfunction is proximal renal tubular acidosis. These data are mostly obtained from clinical studies of individuals with defects in the SLC4A4 gene, genetic linkage studies, and studies of genetically altered mice. I) Central nervous system. A) Mental retardation, migraine, and epilepsy. Many individuals with defects in the SLC4A4 gene present with mental retardation (see TABLE 6). The presence of this trait in individuals with the Q29X mutation, which is predicted to abrogate renal (i.e., NBCe1-A) but not neuronal or glial NBCe1 expression (i.e., NBCe1-B and -C), suggests that mental retardation can be secondary to whole body acidosis that is the signature of pRTA.

In a separate study, genetic-linkage analysis mapped a familial temporal lobe epilepsy (including “déja` vu” auras) to a chromosomal locus that includes SLC4A4, although sequencing of SLC4A4 exons in two affected individuals did not reveal any genetic abnormalities (374). However, the possibility of a linkage remains open as the promoter regions of SLC4A4 were not included in this analysis and it is not disclosed whether the exons of all NBCe1 variants were sequenced. II) Sensory organs. A) Glaucoma, band keratopathy, cataracts, and corneal edema. Many individuals presenting with pRTA have ocular defects ranging from glaucoma and band keratopathy to total blindness (TABLE 6). The widespread expression of NBCe1 throughout the eye as well as the absence of certain ocular phenotypes in some individuals with pRTA indicate that ocular phenotypes need not necessarily be secondary to whole body acidosis. In these patients, it is not clear what causes the high-tension glaucoma, a buildup of aqueous humor that causes an increase in intraocular pressure. The two major forms of glaucoma in the general population are due to decreased disposal of the aqueous humor, ultimately via the trabecular meshwork in the anterior chamber. As noted earlier, NBCe1 has been detected in the trabecular meshwork (989), but the role of NBCe1 in this tissue is untested. Glaucoma is not a feature associated with pRTA in individuals with either the T485S or G486R mutations (TABLE 6). The pathogenesis of cataracts in some individuals with SLC4A4 defects could be caused by defective Na⫹ transport or pHi regulation in lens epithelial cells. Perturbations of either process are associated with increased lens opacity

Band keratopathy, corneal opacity caused by the deposition of Ca2⫹ salt can be secondary to renal failure (briefly reviewed in Ref. 149). In one case, a 12-yr-old girl with a defect only in (renal) NBCe1-A variant did not have band keratopathy (412), consistent with variable penetrance or with the hypothesis that it is specifically a defect in NBCe1-B and -C in the corneal endothelium that causes the band keratopathy. If HCO3⫺ secretion across the corneal endothelium and into the aqueous humor were compromised, a localized elevation of [HCO3⫺] in the subepithelial region of the corneal stroma might be expected to enhance the deposition of Ca2⫹ salts (928, 989). Band keratopathy is not a feature associated with pRTA in individuals with the Q29X or R881C mutations (TABLE 6). The eyes of NBCe1-null mice appear normal. These mice die at 4 wk (313) before any ocular defects are evident. However, in a line of transgenic mice that express the mouse ortholog of the human NBCe1/W516X pRTA mutant (FIGURE 25 and TABLE 6), corneal opacity and edema are evident when these mice are kept alive beyond week 7 by administration of HCO3⫺ (602). III) Peripheral nervous system. We are unaware of any reports of peripheral nervous system dysfunction associated with NBCe1 mutations. IV) Circulatory system. A) Possible contribution to ischemic and reperfusion injury in the heart. As discussed earlier, humans with cerebral artery occlusion, abdominal aorta constriction, or heart failure all appear to have elevated NBCe1 abundance. It is undemonstrated whether ischemic and reperfusion injuries are a cause or consequence of NBCe1 upregulation, but it is possible that elevated NBCe1 levels could increase the risk factor for ischemic injury and heart failure. Indeed, Giffard and co-workers (318) observed that overexpressing NBCe1-B in the 3T3 fibroblast cell line renders these cells susceptible to acid injury in the presence of HCO3⫺, perhaps via Na⫹-loading and thence Ca2⫹-loading via a Na-Ca exchanger (318). Many studies have suggested that NBCe1 inhibitors could be cardioprotective (225, 481, 840). V) Musculoskeletal system. A) Growth retardation. Individuals with defects in NBCe1 typically exhibit below average height and weight (see TABLE 6), and mouse models of NBCe1-associated pRTA exhibit bone dysplasia (313, 602) and reduced muscle mass (602). NBCe1 is not known to be directly involved in bone remodeling, and it is possible that these signs are secondary to the whole body acidosis that accompanies pRTA.

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Migraine is a symptom associated with many different NBCe1 mutations that cause pRTA (930), although the precise molecular mechanism underlying this pathology is unknown. At least in one case of two sisters, it appears that a 65-bp deletion affecting the Ct of all NBCe1 variants is also associated with epilepsy (72, 930).

(65, 240, 732). Cataracts are not a feature associated with pRTA in the individual with the Q29X mutation that is specific to NBCe1-A (TABLE 6).

MARK D. PARKER AND WALTER F. BORON

Table 6. SLC4A4 mutations in individuals with proximal renal tubular acidosis Label in Figure 25

Trivial Name and Original Report

Predicted Protein Producta

DNA

b

Likely Molecular Basis for pRTA

Q29X (412)

p.Gln29X

c.85C⬎T

Protein not translated (929)

2

R298S (411)

p.Arg298Ser

c.894A⬎C

3

S427L (253)

p.Ser427Leu

c.1280C⬎T

Partial mistargeting to apical membrane with some cytosolic retention (929)d combined with a approximately 25% reduction in permolecule function (166). Partial mistargeting to apical membrane (577) combined with a likely reduction in per-molecule function (253, 577).

4

T485S (393)

p.Thr485Ser

c.1453A⬎T

5

G486R (929)

p.Gly486Arg

c.1456G⬎A

6

R510H (411, 879)

p.Arg510His

c.1529G⬎A

7

W516X (602)

p.Trp516X

Not reportedg

Complete loss of protein (602)

8

L522P (241)

p.Leu522Pro

c.1565T⬎C

Intracellular retention of mutant (241, 929, 930)

Mutant traffics normally thus loss of function is likely explained by impaired per-molecule activity (393, 576, 929, 930). Mutant traffics normally;e thus loss of function is likely explained by impaired per-molecule activity (929). Intracellular retention of mutant (577, 930). Additional per-molecule activity defects not reported.f

Mental retardation, growth retardation, glaucoma. No evidence of cataracts or band keratopathy. Mental retardation, growth retardation, glaucoma, cataracts, and band keratopathy. Elevated serum amylase. Calcification of basal ganglia (414).

Growth retardation, glaucoma, and cataracts. Poor dentition. Normal intelligence. No specific mention of band keratopathy, but corneal clouding was evident. Also some evidence of respiratory acidosis (i.e., elevated Pco2). Growth retardation, cataracts, and band keratopathy. No specific mention of mental retardation or glaucoma. Growth retardation, cataracts, and band keratopathy. Normal intelligence and no glaucoma. Growth retardation, glaucoma, cataracts, and band keratopathy. Delayed neurological and motor development. No mention of mental retardation. Migraines (930). Growth retardation, glaucoma, cataracts, and band keratopathy. Calcification of basal ganglia. No mention of mental retardation. Motor and mental retardation, growth retardation, glaucoma, cataracts, and band keratopathy. Dental abnormalities. Migraines.

Continued

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1

Pathological Features From Original Report (Other Than pRTA)c

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Table 6.—Continued Label in Figure 25

Trivial Name and Original Report

Predicted Protein Producta

DNA

Likely Molecular Basis for pRTA

b

p.Asn721ThrfsX30i

c.2162delA

Complete loss of protein (416)

10

A799V (393)

p.Ala799Val

c.2396C⬎T

11

R881C (393)

p.Arg881Cys

c.2641C⬎T

Intracellular accumulation of mutant, reduced per-molecule function, ⫺ and a HCO3 independent conductance (721). Intracellular retention of protein (576, 930, 977, 1113). Protein has close to normal per-molecule activity (977).

12

⌬65bp (413, 930)

p.Ser982AsnfsX4k

c.2944_2967 ⫹ 42dell

Intracellular retention of mutant (930), but mutant protein functions normally when expressed in oocytes (930).

Growth retardation, cataracts, band keratopathy. Dental abnormalities. Calcification of basal ganglia. Normal intelligence. No glaucoma, but elevated ocular pressure. Elevated serum lipase and amylase.j Motor and mental retardation, growth retardation, low weight, glaucoma, cataracts, band keratopathy, and calcification of basal ganglia (231). Growth retardation, glaucoma, cataracts. No specific mention of mental retardation or band keratopathy. Elevated serum amylase. Migraines (930). Glaucoma, cataracts, and band keratopathy. Migraines. Normal intelligence and stature. Epilepsy. Nausea.

A database of human SLC4A4 mutations is curated at the Leiden Open Variation Database (https:// grenada.lumc.nl/LOVD2/shared1/home.php?select_db⫽SLC4A4). The position of the mutated residues within NBCe1 is depicted in Figure 25 and shown on sequence alignments in Appendix I. aBased on NP_003750. bBased on NM_003759.3 (“A” of initiating ATG codon is counted as nucleotide 1). cFeatures not described in the original report are provided together with a reference to the paper in which the feature was described. dReports conflict as to whether the equivalent mutation in NBCe1-B (p.Arg324Ser) causes the protein to be mistargeted. The mutant is reported to accumulate normally in the plasma membrane of a human-bladder-endothelium cell line (836) and a rat glioma cell line (930) but to be retained in the cytosol of a canine-kidney-epithelium cell line (577). Interestingly, an artifical mutant—R298C— created in a version of NBCe1-A that lacks the five endogenous, cytoplasmic cysteine residues traffics efficiently to the plasma membrane in a human-kidney-epithelium cell line (1113). eIn a rat glioma cell line, the equivalent mutant of NBCe1-B (p.G530R) appears to have an increased intracellular presence compared with the wild-type transporter (930). fOne study finds that the equivalent mutation in NBCe1-B (p.Arg554His) exhibits a loss of function but not reduced accumulation of transport protein in the plasma membrane of human endothelial cell line (836). gLikely c.1547G⬎A or c.1548G⬎A. hThis designation counts the first base of the 5=-UTR as nucleotide 1. iFirst affected amino acid is Asn721, which changed to Thr. Thr becomes residue #1 of the frame shifted reading frame (fs) that has a termination codon at position #30. The unique 29-amino acid appendage is predicted to have the sequence TEVGSFHRLEKTPGGCALLLLSRLCWSLY. jThe authors mention only elevated lipase in their clinical description of the patient, but refer also to elevated amylase in the discussion of their findings. kThe unique 3-amino acid appendage is NKF (930). l2944 –2967 of the exon 23 are missing plus 42 of the following intron.

B) Hypokalemic paralysis. An individual with the A799V mutation in NBCe1 exhibited hypokalemic paraplegia (231). It is notable that the mutant NBCe1, when expressed in Xenopus oocytes exhibits a HCO3⫺-independent ion leak (721). Such a leak could in principle contribute towards the observed paralysis, as has been suggested in the case of unusual ion leaks though Na⫹ and Ca2⫹ channels (293, 1047). Under hypokalemic conditions, the conductance of inward-rectifier K⫹

channels in muscle cells is reduced, destabilizing their membrane potential. In this situation, small pathological currents can make a disproportionately large contribution to resting Vm, and the prolonged depolarization of the cells that results, inactivates voltage-gated Na⫹ channels (820). VI) Upper digestive system. A) Compromised enamel deposition. The teeth of humans with a mutant SLC4A4 gene

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nt2311h (416)

9

Pathological Features From Original Report (Other Than pRTA)c

MARK D. PARKER AND WALTER F. BORON

NBCe1-A

EL3 9 6 7 8

1

2

4/5

3

4

Lumen

5

6

7

8

9 10

10

11

E 13

14

3

Cytosol

11 2

Ct

Nt

H2N

12

1

FIGURE 25. Location of disease-associated mutations in NBCe1-A. Representation of human NBCe1-A topology from FIGURE 2A. Numbered circles show the positions of SLC4A4 mutations (numbers 1–12 match the numbered mutant descriptions in TABLE 6) that cause proximal renal tubular acidosis. Nonsense mutations that result in premature translational termination are colored red, and missense mutations are colored green. (TABLE 6),

and of mice with a disrupted Slc4a4 gene (313, 538), exhibit signs of defective enamel deposition. NBCe1 is expressed in the enamel organ (456, 538, 706), and CO32⫺ is an important constituent of enamel (reviewed in Ref. 564). However, the precise role of NBCe1 in the process of enamel remodeling (see FIGURE 20) has yet to be elucidated. It is possible that the defect is not secondary to whole body acidosis, inasmuch as unusual dentition was not noted in the description of a 12-yr-old girl who was predicted to lack only the renal variant of NBCe1 and unaffected ameloblastic NBCe1 (412). Furthermore, the defect is unlikely to be secondary to a reduced salivary pH because, in mice, the defect is noted in preerupted teeth (538). The likely importance of NBCe1 for correct enamel deposition was recently reviewed by Urzúa et al. (988). VII) Lower digestive system. A) Intestinal obstruction. Although individuals with SLC4A4 defects are not reported to have intestinal problems, genetic linkage analysis has indicated that NBCe1 could contribute to the severity of ileal obstruction in newborns with cystic fibrosis (257). Indeed, NBCe1-null mice have small ceca and those that survive beyond 20 days have impacted terminal ilea, ceca, and colons (313), a phenomena also reported in the W516X mouse model of pRTA (602). However, the defective net ion secretion that is observed in isolated colons from NBCe1-null mice could be explained by increased fluid absorption due to dysregulation of other ion transporters such as ENaC and NKCC, rather than by decreased fluid secretion due to a lack of NBCe1 per se (313). B) Possible signs of pancreatitis. The abundance of NBCe1-B in the pancreas suggests that this protein plays a major role

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in HCO3⫺, and consequently fluid, secretion by this organ. Dysfunction of NBCe1-B, like dysfunction of CFTR (e.g., Ref. 845), might therefore result in pancreatitis. Although some individuals with NBCe1-associated pRTA exhibit molecular signs of pancreas dysfunction, such as elevated serum amylase and lipase (TABLE 6), clinical signs of pancreatitis have not been reported. NBCe1-null mice have a normal pancreatic histopathology, although, as the authors of that study note, these young mice51 may have been examined prior to development of pancreatic pathology. In addition, the mouse pancreas is known to be a poor model for human pancreatic insufficiency (313). Taken together, these observations suggest that development of pancreatitis due to NBCe1 dysfunction may be age-dependent, or prevented by an as-yet-unidentified mechanism. For example, NBCn1 could compensate for NBCe1 loss in the basolateral membranes of exocrine duct cells (FIGURE 21). C) Potential contribution to diarrhea. In the colon, NBCe1 action could support HCO3⫺ secretion and thereby promote Cl⫺ absorption via the apical Cl-HCO3 exchanger Slc26a3 (DRA, see Ref. 388) and the basolateral chloride channel ClC-2 (see Ref. 159). If this hypothesis is correct, then NBCe1 dysfunction is expected to reduce fluid absorption and promote diarrhea. However, the colons of NBCe1-null mice do not exhibit fluid absorption defects perhaps due to the documented dysregulation of other ion transporters in the colons of these mice (313).

51

NBCe1 null-mice rarely survive beyond weaning age (313).

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HOOC

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

VIII) Lymphatic and immune systems. Mice with a disrupted Slc4a4 gene exhibit severe splenomegaly with an increased count of nucleated red blood cells (313, 602). However, the spleen is not a major site of NBCe1 expression, and both phenomena are considered to be secondary to whole body acidosis (313, 602).

B) Hypertension. Two independent genome-wide association studies reported in Reference 1077 link the SLC4A4 gene locus with hypertension in individuals from China. However, the precise location of hypertension-associated markers within SLC4A4 is not stated. Mice that lack aromatic amino acid decarboxylase (AADC) in their PTs develop salt-sensitive hypertension. The kidneys of these mice exhibit elevated mRNA abundance of a number of transporters involved in Na⫹ reabsorption, including NBCe1 mRNA (1094). However, because of the complex effects of AADC disruption, the specific contribution of NBCe1 to hypertension in these mice is difficult to assess. It is noteworthy that one strain of spontaneously hypertensive rat exhibits elevated renal NBCe1 abundance (900). X) Reproductive system. A) Expected, but not demonstrated, reduction in fertility. Based on the putative role of NBCe1 in the reproductive tracts of males (i.e., maintenance and activation of sperm) and females (i.e., enhancing sperm fertilizing capacity), it is possible that NBCe1 defects could be associated with a loss of fertility. However, this remains unverified as Slc4a4-null mice do not survive to

2. NBCe2 (Slc4a5) A) SUMMARY. The electrogenic Na/HCO3 cotransporter NBCe2 (encoded by the Slc4a5 gene) is present in many organ systems throughout the body but is notably abundant in the choroid plexus, where NBCe2 contributes towards HCO3⫺/fluid (i.e., CSF) secretion into the brain ventricles, and the liver, where robust expression of NBCe2 likely maintains hepatocyte pHi.

Consistent with its proposed role in the choroid plexus, one strain of Slc4a5-null mouse exhibits a CSF secretion defect that likely contributes to the reduced neuronal excitability observed in these animals. In humans, multiple studies report genetic linkage between the SLC4A5 locus and blood pressure traits. Although the physiopathology underlying this linkage is presently unclear, Slc4a5-null mice exhibit elevated blood pressure. Little is known about the regulation of the Slc4a5 gene or products and is the only one of the five NCBTs that has not been demonstrated to be stimulated by the cytosolic protein IRBIT. NBCe2 has only one variant, NBCe2-c, that is known to be functional. B) NOMENCLATURE OF Slc4a5 PRODUCTS. The Slc4a5 gene product was initially called NBC4 (767), being the fourth member of the gene family to be identified at the molecular level. The gene product has since been renamed NBCe2 (1009) to reflect its characterization as the second electrogenic member of the family (835, 1009).

Six variant products have been reported (NBC4a-f), but only two (NBC4a and NBC4c) seem likely to produce a functional transporter. We provisionally refer to these as NBCe2-a and NBCe2-c (note the lowercase “a” and “c”). C) MOLECULAR ACTION OF NBCe2. Overexpressed in HEK-293T

or mPCT renal cells (835) and Xenopus oocytes (1009), NBCe2-c mediates a reversible, DIDS-sensitive, and Na⫹dependent HCO3⫺ transport that is accompanied by a Na⫹and HCO3⫺-dependent conductance. Thus NBCe2 is an electrogenic Na/HCO3 cotransporter (FIGURE 16). Furthermore, in oocytes, NBCe2-mediated HCO3⫺ efflux does not require extracellular Cl⫺ and thus NBCe2 is not a Na⫹driven Cl-HCO3 exchanger (1009). In mPCT cells (a mouse PT cell line) that are overexpressing NBCe2 (835), and in mouse choroid plexus epithelia (645), NBCe2 operates with a Na⫹:HCO3⫺ stoichiometry of 1:3. However, when heterologously expressed in oocytes (1009) or HEK-293 cells (869), NBCe2 operates with a 1:2 stoichiometry. This celldependent stoichiometry is also characteristic of NBCe1. As well as being blocked by DIDS, NBCe2 is inhibited by the NBCe1 blocker tenidap, as demonstrated for NBCe2 expressed in HEK-293 cells (869).

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IX) Urinary system. A) Proximal renal tubular acidosis. Twelve homozygous mutations have been described in the SLC4A4 gene in individuals with proximal renal tubular acidosis (pRTA; see TABLE 6 AND FIGURE 25). As expected, considering the role of NBCe1 in the kidney, pRTA is characterized by an impaired ability of the PT epithelium to reabsorb HCO3⫺, leading to a whole body metabolic acidosis. In individuals with defective NBCe1, plasma pH (7.08 – 7.23) and plasma [HCO3⫺] (5.6 –15 mM) are both below the normal range. A genetic defect specific to NBCe1-A “Q29X” (FIGURE 25 AND TABLE 6) is predicted to produce no functional NBCe1-A transporter, but not to affect the production or activity of either the NBCe1-B or -C variants (412). Two further nonsense and nine missense mutations have also been identified in the SLC4A4 gene in individuals with pRTA (see TABLE 6). The genetic linkage between defective NBCe1 and pRTA is further strengthened by the demonstration of whole body acidosis in Slc4a4-null mice, which die shortly after weaning (313), and in a transgenic mouse model of pRTA that carries the human NBCe1/W516X pRTA-associated mutation (602). It is likely that at least some of the nonrenal sequelae associated with pRTA, such as retarded growth and mental retardation, may in part be secondary to the whole-body metabolic acidosis.

breeding age (313) and the fertility of humans with NBCe1associated pRTA remains unreported.

MARK D. PARKER AND WALTER F. BORON D) THE SLC4A5 GENE. The human NBCe2 gene maps to chromosomal locus 2p13(FIGURE 26A and Ref. 767) and has at least 31 exons that encompass ⬃127 kb of genomic DNA (FIGURE 26B). Reports that the 5=-UTR of NBCe2 includes sequence from exons shared with its neighboring gene DCTN1 (770), which encodes the p150GLUED subunit of dynactin, appear to have been premature. The presently assigned SLC4A5 and DCTN1 gene boundaries are separated by ⬃15 kb (NCBI human genome assembly 37 version 1) and at least two promoters for SLC4A5 transcription are located within this intergenic region (916). The initiator codon for all presently known variants of NBCe2 is located in exon 6 of SLC4A5. One SLC4A5 promoter regions is located upstream of exon 1, but transcripts most often exclude exon 1 and start at exon 2, and promotes robust expression of a reporter gene in a humanlung and a mouse-myoblastoma cell line, but not in a human embryonic kidney cell line. The second SLC4A5 promoter is upstream of exon 5 and promotes robust expression of a reporter gene in the lung and kidney cell lines, but not in the myoblastoma cell line (916).

a/NBC4a and NBCe2-c/NBC4c are predicted to encode a functional transporter, NBCe2-c being the more abundant of the two. GenBank protein accession numbers for the variants discussed in this section are provided in Appendix IV. I) Sources of variation in coding sequence among NBCe2 variants. Unusually for an NCBT, the Nt and Ct sequences of NBCe2 are not known to be variant, although in silico analysis suggests the existence of yet-to-be-reported gene products. As there are only two validated variants of NBCe2, there is only one validated source of variation that distinguishes NBCe2-a from NBCe2-c.

E) STRUCTURAL FEATURES AND VARIANTS OF NBCe2. Of the six originally reported splice variants of NBCe2 (NBC4a-f) (768, 770, 835, 1009, 1056), the cDNA sequences of only four (NBC4a-d) match the human genome. Only NBCe2-

A

52 For example, baboon (XP_003908879), gibbon (XP_003268733), and orangutan (XP_003775944).

Locus 2p13 20 kb

MTHFD2

B

C

DCTN1

Gene structure

P1

12

SLC4A5

10 kb

P2

3

5

27

6

Transcript variation P1

P2 M

NBCe2-a

1

2

3

4

5

NBCe2-c

1

2

3

4

5

6

26

6

26

27

28

29

30

* 31

32

28

29

30

* 31

32

M

FIGURE 26. SLC4A5 gene structure and NBCe2 transcript variants. Scale diagrams showing the human SLC4A5 gene locus together with the position of neighboring genes (A), the position of promoters (P1, P2, and P3), and the position of exons within SLC4A5 (B). Transcript variants are represented, not to scale, as numbered boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature transcript. “//” denotes that both transcripts include exons 6 –26. Exons that include the initiator ATG codon (“M”) and termination codon (“*”) are marked for each transcript. Colored exons, or parts of exons, correspond to the protein regions that each encodes, which are identically colored in FIGURE 27. Uncolored exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line are predicted, but not demonstrated, to be included in the mRNA. Not shown are NBC4b and NBC4d that are unlikely to encode stable/functional transporters, or NBC4e and NBC4f that are cloning artifacts (see text).

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A) Extension between putative TMs 11 and 13. Transcripts that encode NBCe2-a include exon 27 (FIGURE 26C). The inclusion of the novel exon is predicted to lengthen, by the 16-amino acid sequence “MGTGGSEFKIQKKLTP,” the predicted extended structure between putative TMs 11 and 13, which includes an intracellular loop (FIGURE 27). This extension, also predicted to be included in NBCe2 variants from various primates,52 includes

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Nt

TMD 100 aa

1–5

6–9

Ct 10–14 1,137

NBCe2-a 16

1,121

NBCe2-c

FIGURE 27. NBCe2 protein variants. Scale diagram of protein variants that are encoded by the transcripts represented in FIGURE 26C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino acids and colored to denote their genetic origin as shown in FIGURE 26C. A color-matched protein sequence alignment of the variants is provided in Appendix V.

II) Cloned NBCe2 variants that are demonstrated or likely to exhibit NCBT activity. A) NBCe2-a/NBC4a (NCBT activity untested). NBCe2-a is the longest NBCe2 variant, as it includes the 16-amino acid splice cassette mentioned above (FIGURE 27). We regard NBCe2-a as likely to exhibit NCBT activity because we have no evidence that the 16-amino acid insertion would disrupt function. B) NBCe2-c/NBC4c (NCBT activity demonstrated). This remains the only variant that has been functionally characterized (835, 1009). NBCe2-c lacks the 16-amino acid insertion found in NBCe2-a (FIGURE 27) and is therefore structurally most similar to other NCBTs within its TMD. III) Predicted NBCe2 variants. Based on our in silico analysis, the alternative splicing of NBCe2 RNA has the potential to generate a novel protein variant (i.e., not NBC4a–f). Excision of exon 30 would produce an NBCe2 variant in which the last 39 amino acid of NBCe2-c are replaced with an alternative 77-amino acid Ct appendage that, like the 61-amino acid Ct appendage of NBCe1-C, is predicted to terminate with the class I PDZ-binding domain motif “ETTL.” However, such transcripts have yet to be amplified from mammalian cDNA.

C) NBC4e and NBC4f (probable cloning artifacts). The nucleotide sequences of NBC4e and NBC4f (1056) poorly match the human genome and contain frame-shifts and nonsense mutations (producing premature termination) that distinguish them as being artifactual. At least as expressed in oocytes, NBC4e, which has a truncated Ct, was reported to mediate a substantial pHi recovery from a CO2/ HCO3⫺–induced acid-load. In separate experiments on the same cell, Vm was unperturbed by the application of CO2/ HCO3⫺ (1056). Taken at face value, these data suggest that the peculiarities of the NBC4e construct compromise its electrogenicity. DIDS sensitivity, Na⫹ dependence, and HCO3⫺ dependence of the pHi recovery mechanism were not examined in these experiments. F) DISTRIBUTION OF NBCe2. The major organ most often associated with NBCe2 expression is the liver, although NBCe2 is expressed in many other organs. The distribution of NBCe2 in specific organ systems is discussed below. The distribution of NBCe2 is summarized and compared with that of other NCBTs in TABLE 5.

IV) Other NBCe2 variants. A) NBC4b (potentially legitimate transcript, NCBT activity unlikely). NBC4b is identical to NBCe2-a except for the presence of a 16-nt exon (768) that produces a frame-shift, causing the last 8 amino acids of putative TM14 as well as the entirety of the 83 amino acids in the Ct to be replaced by 28 novel amino acids. Especially with the changes to TM14, it is not clear that the protein would be stable or functional.

I) Central nervous system. A) Blood-brain barrier and elsewhere. RT-PCR analysis reveals the presence of NBCe2 transcripts in human brain (214, 835), specifically in the choroid plexus epithelium (CPE), hippocampus, cerebrum, and cerebellum (214) and in the hippocampi of mice (73). Western blotting and immunohistochemical studies localize NBCe2 protein to the apical membranes of rat and mouse CPE (113, 470), see cartoon in FIGURE 28. Immunogold staining confirms the presence of NBCe2 protein in the membranes of apical microvilli of mouse CPE (113). Interestingly, human CPE is not labeled by existing anti-NBCe2 antibodies (113), despite the presence of NBCe2 transcripts in this choroid-plexus preparation.

B) NBC4d (potentially legitimate transcript, NCBT activity unlikely). NBC4d (770) lacks sequence encoding TMs 11–13 and is thus unlikely to encode a functional transporter.

II) Sensory organs. A) Eye. Analysis of EST abundance suggests that NBCe2 is expressed in the human and mouse eye (Appendix VI). In the retina, NBCe2 protein is located in the outer plexiform layer (470).

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a consensus PKA phosphorylation site. The functional consequences of the splicing of this cassette are unknown.

MARK D. PARKER AND WALTER F. BORON

CSF

Tight junction

Interstitial space

H2O 2 K+

Na-K pump 3

Cl– HCO3–

Na+ H+

K+

KCC4

Na+ HCO3–

K+ Cl–

++

cAMP

H+

NHE

CA

NBCe2 3 HCO3–

NBCn1

HCO3–

CO2 H2O

Na+ Cl–

NBCn2

HCO3– Na+

Na+

K+ 2

Cl–

Cl–

NDCBE

–

2 HCO3

Choroid plexus epithelium FIGURE 28. Role of NCBTs in the choroid plexus. The secretion of cerebrospinal fluid by the choroid plexus (CP) and intracellular pH regulation in CP epithelia is achieved by a combination of NCBTs. NBCe2 is the major ⫺ NCBT that is responsible for HCO3 secretion across the apical membrane. NBCn1, NDCBE, and NBCn2 have ⫺ all been detected in the basolateral membrane of CP epithelia, mediating HCO3 influx. NBCn1 has also been detected in the apical membrane of the CP in some strains of mice. Note that NDCBE is not present in the CP of adults. Reported, but not shown, is the presence of NBCe1 in the basolateral membrane.

III) Peripheral nervous system. A) Trigeminal ganglion. NBCe2 transcripts are detected by RT-PCR in preparations of rat trigeminal ganglion neurons (408). IV) Respiratory system. A) Lungs. Northern blot analysis reveals the presence of NBCe2 transcripts in a human lung preparation (767, 768). Western blotting and immunohistochemical studies localize NBCe2 protein to the basolateral membranes of a human airway epithelial cell line (515). V) Circulatory system. A) Heart. NBCe2 transcripts have been detected in heart preparations from humans (767, 768), mice (31), and rats (1056). Indeed, the archetypa human NBC4a transcript was cloned from heart cDNA (768). Analysis by qPCR reveals NBCe2 transcripts in mouse ventricles at a similar abundance to other cardiac HCO3⫺ transporters such as AE3 and NBCe1 (31). A preliminary immunocytochemical study on rat ventricular myocytes suggests that NBCe1 protein is more abundant than NBCe2 in these cells (311).

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VI) Musculoskeletal system. A) Muscles. RT-PCR reveals the presence of NBCe2 transcripts in a human muscle preparation (835). NBCe2 protein is detected in rat and human muscle homogenates (518). In rat muscle, NBCe2 is predominantly localized to the sarcolemma with an additional presence in T tubules (518). VII) Upper digestive system. A) Stomach. Northern blot analysis reveals the presence of NBCe2 cRNA in a human stomach preparation (767, 768). VIII) Lower digestive system. A) Widespread, abundant in liver. The liver appears to be one of the major sites of expression of NBCe2 transcripts (767, 768, 1056). A western blot and immunohistochemical study have localized NBCe2 protein to the basolateral (i.e., sinusoidal) membrane of rat hepatocytes (FIGURE 29) and to the apical membrane of cholangiocytes in rat intrahepatic bile ducts (8). Northern blots reveal the presence of NBCe2 transcripts in

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Na+

Na+

NKCC1

AE2

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

H+ Interstitial space

NHE1 ++

NBCe2 K+

Na+ Na+

cAMP

2 HCO3–

Hepatocyte

H+ HCO3–

Cl–

CAII

HCO3–

AE2

CO2 H2O

CFTR Cl–

2 K+

Na-K pump

3 Na+

⫺ FIGURE 29. Role of NBCe2 in hepatocytes. NBCe2 mediates HCO3 influx across the basolateral membrane ⫺ of hepatocytes. This activity regulates intracellular pH and supports AE2-mediated HCO3 secretion into the bile ducts.

human small intestines (767, 768), a distribution that was determined by RT-PCR to correspond to NBCe2 expression in at least ileum, jejunum, and duodenum (214). Elsewhere, NBCe2 transcripts have also been detected in preparations of human pancreas (835) and proximal and distal colon of rodents (512, 1056).53 However, in mouse duodenum, jejunum, ileum, and colon, the abundance of NBCe2 mRNA is trivial compared with the abundance of NBCe1 or NBCn1 mRNAs (180). Accordingly, the duodena of NBCe2-null mice exhibit no detectable HCO3⫺ secretion defects (180). 53

It has been noted by Odgaard et al. (694) that the NBCe2specific primers reported by Xu et al. are not derived from NBCe2 sequence. This appears to have been an errant description of the primer sequences in the paper rather than in the design of the actual primers used by Xu et al. that were GCCAGCTATGCATGAAATTG (sense) and ATGGGTCCTGTGCTGCTGAG (antisense; J. Xu and M. Soleimani, personal communication).

IX) Lymphatic and immune systems. A) Spleen and leukocytes. NBCe2 transcripts are have been detected in preparations of human spleen (767, 768) and peripheral blood leukocytes (835). X) Endocrine system. A) Thyroid. In situ hybridization experiments reveal the presence of NBCe1 transcripts in the thyroid glands of 1-day-old and adult mice (341).54 Analysis of the abundance of ESTs suggests that NBCe2 is expressed in the human thyroid and parathyroid glands (Appendix VI). XI) Urinary system. A) Kidney. NBCe2 transcripts have been detected in human kidney preparations (767, 768), 54 Despite the abundance of NBCe2 transcripts in the mouse thyroid, the morphology and serum T4 abundance were normal in NBCe2-null mice (341).

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Canaliculus

MARK D. PARKER AND WALTER F. BORON corresponding to the presence of NBCe2 transcripts in both the kidney cortex and medulla (214, 341, 1056). Within the medulla, NBCe2 transcripts have been detected in the outer medullary segments of the TAL of rats (1056), and in IMCD of humans (986) and mice (341). Immunohistochemical studies suggest an apical localization of NBCe2 protein in outer medullary collecting duct cells of humans (214), and in uroepithelial cells of the renal pelvis of rats (8). XII) Reproductive system. A) Male. NBCe2 transcripts have been detected in preparations of human testis (767, 768, 835) and in mouse testis, epididymis, and vas deferens (599). The archetypa clones of NBC4b (767, 768), NBC4c (835), and NBC4d (835) originate from human testicular cDNA.

C) Placenta. NBCe2 transcripts have been detected by northern blot of human placenta RNA (767, 768). G) PHYSIOLOGICAL ROLES OF NBCe2. Despite the broad distribution

of NBCe2 throughout the body, few physiological roles have been ascribed to NBCe2 action. Aside from its roles in the choroid plexus and liver, described below, NBCe2 likely contributes to pHi regulation in all of the cell types in which it is located. Further physiological roles are suggested by the signs exhibited by NBCe2-null mice, although primary and secondary effects of NBCe2 loss have yet to be distinguished. I) Central nervous system. A) Support of CSF secretion. In the choroid plexus epithelium, which is basically a backwards proximal tubule, the apical membrane faces the CSF. Thus apical NBCe2 (113), which appears to operate with a 1:3 stoichiometry (645), would be in a position to mediate the apical step (i.e., HCO3⫺ efflux) of HCO3⫺ secretion into the CSF (FIGURE 28). Other NCBTs such as NBCn2, and in some instances NBCn1 and NDCBE, are present at the basolateral (blood-side) membrane of CPE, and presumably mediate the basolateral (i.e., HCO3⫺ uptake) step of HCO3⫺ secretion. The role of NBCe2 in proper CSF secretion is supported by the exhibition of defective CSF secretion in NBCe2-gene-trapped mice, although the CPE in these mice exhibit other defects. Furthermore, ventricle size is normal in a different strain of NBCe2-null mice (341). II) Peripheral nervous system. A) Potential contribution to neuronal excitability. NBCe2 in cultured rat trigeminal ganglion neurons could contribute towards countering the excitability-dampening effects of intracellular acidification, although NBCe1-B/C appears to be the dominant DIDSsensitive Na/HCO3 transporter in these cells (408). III) Lower digestive system. A) pHi regulation in hepatocytes. The basolateral location of hepatocellular NBCe2 (FIGURE 29) is consistent with the previously demonstrated

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B) [Na⫹]i regulation in hepatocytes. The addition of CO2/ HCO3⫺ to primary cultures of rat hepatocytes causes a substantial increase in hepatocellular [Na⫹]i. The increase in [Na⫹]i accompanying NBCe2-mediated HCO3⫺ influx has a substantial stimulatory effect on the activity of the Na-K pump (288). Moreover, in perfused livers, the addition of CO2/HCO3⫺ increases O2 consumption. These data are consistent with the hypothesis that a Na/HCO3 cotransporter, most likely NBCe2, mediates a substantial Na⫹ influx, which in turn increases the demand on the Na pump, and thus on oxidative metabolism. C) Choliangiocyte viability. The application of the NCBT inhibitor S3705 inhibits the growth of and promotes apoptosis in cholangiocarcinoma cells (247), perhaps in part by inhibition of cholangiocyte-expressed NBCe2. D) Potential contribution to transepithelial HCO3– transport in cholangiocytes. Na/HCO3 cotransport, most likely mediated by NBCe2, may constitute a basolateral step (i.e., HCO3⫺ uptake) in secretion of HCO3⫺ into the hepatic bile canaliculi (60, 88). However, in cholangiocytes lining the bile ducts of rats, NBCe2 apparently has an apical distribution (8). Considering that a major role of the bile duct is to secrete HCO3⫺ into the lumen, and that the accepted major pathway for apical HCO3⫺ exit from rat cholangiocytes is Cl-HCO3 exchange mediated by AE2 (46, 59, 631, 902, 987), the role of an apical NBCe2 in these cells is unclear. The cholangiocytes of mice express NBCe1 (987). In these animals, NBCe1 may compensate for AE2 insufficiency (987). It is possible that NBCe2 plays a similar support role in the cholangiocytes of rats. IV) Urinary system. A) Possible role in HCO3– reabsorption. Because NBCe2-null mice exhibit signs of urinary HCO3⫺ wasting, it has been suggested that NBCe2 might normally contribute towards renal HCO3⫺ reabsorption

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B) Female. NBCe2 transcripts have been detected by RTPCR of mouse ovary, uterus, and vagina (599).

presence of an electrogenic NCBT activity in the basolateral membranes of hepatocytes (291, 792). The influx of Na⫹ and HCO3⫺, presumably in a 1:2 stoichiometry, mediated by the transporter under basal conditions in vivo (288, 291) plays a major role in regulating hepatocellular pHi (266, 289, 326) and, as discerned by studies of perfused rat liver, is the primary mechanism of pHi regulation in the intact liver under physiological conditions (266). The maintenance of hepatocyte pHi within a narrow range is crucial for the functioning of the diverse cellular processes such as gluconeogenesis, biotransformation of xenobiotics, and mitogenesis (reviewed in Ref. 287). NBCe2 is cooperatively regulated by the activity of a pH-dependent K⫹ conductance pathway (gK), such that 1) a decrease in pHi causes a downregulation of gK and depolarizes the plasma membrane (presumably reflecting the decrease in gK) and 2) the depolarization stimulates NBCe2 activity and causes a compensatory increase in pHi (290).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

(341). However, as noted below, this phenotype could reflect enhanced HCO3⫺ secretion. H) CAUSES OF NBCe2 UPREGULATION.

To date, only two groups have reported a maneuver that upregulates NBCe2.

I) General. Increased transcript abundance in response to HDAC and methyltransferase inhibitors. NBCe2 transcript levels are increased in an adenocarcinoma cell line after treatment with inhibitors of histone deacetylase and DNA methyltransferase, evidence for the specific regulation of NBCe2 gene expression by epigenetic factors (478).

I) CAUSES OF NBCe2 DOWNREGULATION.

I) Central nervous system. A) Decreased transcript abundance in brain of a mouse model of drug-responsive depression. Mice that become indifferent to rewards after exposure to chronic mild stress are a model for anhedonia, a symptom of depression. A subset of those mice in which such behavior is induced are responsive to treatment with the antidepressant escitalopram. NBCe2 transcripts are twofold lower in the hippocampi of mice that are responsive to escitalopram treatment compared with NBCe2 transcript levels in the hippocampi of nonresponsive mice (73). The physiological relevance of this finding is unclear, although the finding is consistent with a possible link between NBCe2 and drug resistance.

J) CONSEQUENCES OF NBCe2 DYSFUNCTION.

Despite its abundance in the liver, and presumed importance for hepatic function, NBCe2-null mice have no reported hepatic phenotype. In an early report, NBCe2 was considered as a candidate gene for the neurodegenerative and metabolic disease Alström syndrome (767), which maps to the same genetic locus as NBCe2 (i.e., 2p13). However, subsequent work has shown that mutations in the ALMS1 gene on 2p13 cause this syndrome (197, 373).

I) Central nervous system. A) Defective CSF secretion in mice with a disrupted Slc4a5 gene.55 As expected by com-

55 As the authors of this study note, the Slc4a5 gene of these mice is disrupted in the third extracellular loop, and it cannot be discounted that the phenotypes observed in these mice are due to the expression of misfolded Slc4a5 product in Slc4a5-expressing cells. Thus these mice may be a better model of the effects of mutations that cause NBCe2 to misfold, rather than a model for drawing inferences about the physiological roles of NBCe2.

B) Decreased neuronal excitability in mice with a disrupted Slc4a5 gene. Slc4a5– gene-trapped mice have a reduced sensitivity to the proconvulsive drug pentylenetetrazol, a finding that the authors of that study interpret as either a consequence of the reduced intracranial pressure of these mice or of the increased [K⫹]/decreased [HCO3⫺] that is characteristic of the CSF of these mice (470). As NBCe2 has not been demonstrated to be expressed in neurons or glia, it is unlikely that defective pHi regulation in these cells could explain the decreased neuronal excitability, as is thought to underlie a similar resistance in NBCn2-null mice. C) Retinal abnormalities and detachment in mice with a disrupted Slc4a5 gene. Among the diverse morphological abnormalities in Slc4a5– gene-trapped mice, these mice have impaired vision and detached retinas (470). The authors of that study suggest that at least some of these signs may be related to the decreased intracranial pressure in these mice. II) Circulatory system. A) Blood pressure-related traits. Genetic analysis links single nucleotide polymorphisms (SNPs) in the SLC4A5 gene locus with hypertension and other blood pressure-related traits (61, 404, 619, 915, 953, 954), including peripheral artery disease (472). One study reports that the contribution of a particular SLC4A5 SNP to elevated systolic blood pressure in African-American women is greater in individuals with dark versus medium skin color (955). A preliminary report suggests that (SNPs) in the SLC4A5 gene locus are also associated with the relative thickness of the left ventricular wall in hypertensive African-Americans (924). NBCe2-null-mice exhibit elevated blood pressure compared with wild-type littermates (341), although the expression of other hypertension-linked genes (e.g., Slc4a7; see p.

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II) Lower digestive system. A) Increased transcript abundance in colon of mice treated with probiotics. Probiotic treatment of mice is a model used to investigate the molecular mechanisms that underlie the pathogenesis of inflammatory bowel disorder, ulcerative colitis, and the health benefits associated with probiotic treatment. NBCe2 transcript abundance is doubled in the mouse colon 20 days after a probiotic treatment (512).

parison with mice that lack NBCn2, another NCBT that is expressed in CPE and that contributes to CSF secretion, one strain of NBCe2-deficient mice exhibits defects in CSF secretion and thus a decreased brain ventricle volume and intracranial pressure (470). Unexpectedly however, these Slc4a5– gene-trapped mice also exhibit in their CPE, among other defects, a partial redistribution of NBCn2 into the apical membrane, a partial redistribution of the Na/K pump ␣1 subunit into the basolateral membrane, and a complete loss of the Na pump ␤2 subunit (470). Because all of these changes are predicted to exacerbate the CSF secretion defect, we cannot ascribe the decreased CSF secretion solely to the loss of NBCe2 activity per se. CSF composition is also perturbed in these mice inasmuch as it is deficient in HCO3⫺ (but not Na⫹) but contains an overabundance of K⫹ (470). In a second strain of NBCe2-deficient mouse, no decrease in brain ventricle size was observed, although the presence of compensatory mechanisms was not examined in these mice (341).

MARK D. PARKER AND WALTER F. BORON 113) is upregulated in these mice (341). The molecular basis for the observed hypertension in NBCe2-null mice is unknown.

B) Polyuria. NBCe2-null mice exhibit polyuria and polydipsia, although urine osmolality is normal (341).

B. Mammalian Electroneutral NCBTs: NBCn1, NDCBE, and NBCn2 Three of the five mammalian NCBTs perform electroneutral Na⫹-coupled HCO3⫺ transport: NBCn1, NDCBE, and NBCn2. These three transporters are encoded by a group of three closely related Slc4 genes (Slc4a7, -a8, and -a10) that is distinct from two gene groups that encode electrogenic NCBTs or AEs. The three electroneutral transporters appear to have somewhat overlapping distributions, all being abundantly expressed in the CNS where they contribute to enhancing neuronal excitability. As we shall see, the three transporters differ most in their molecular actions. The physiological relevance of these differences is unknown, but these transporters all have the ability to regulate pHi without affecting or being influenced by Vm. 1. NBCn1 (Slc4a7) A) SUMMARY. The electroneutral Na/HCO3 cotransporter NBCn1 (encoded by the Slc4a7 gene) is unique among NCBTs in its low sensitivity to blockade by DIDS as well as in having a HCO3⫺-independent conductance. NBCn1 is unique among electroneutral NCBTs because it does not transport Cl⫺. NBCn1 has the greatest known multiplicity of products for an Slc4 family member (at least 12 known variants, -A through -L) and is present in many organs/ organ systems throughout the body. In common with NBCe1-B/C, NDCBE, and NBCn2, NBCn1 is stimulated by the soluble protein IRBIT. NBCn1 is notably abundant in 1) the central nervous system, where NBCn1 contributes towards neuronal excitability; 2) the eye and the ear; 3) secretory epithelia, where NBCn1 contributes towards transepithelial HCO3⫺/fluid movement (e.g., in the intes-

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B) NOMENCLATURE OF Slc4a7 PRODUCTS. The first report of what we now know as NBCn1 was a partial cDNA, called SBC2 or hNBC2, from a human retinal cDNA library (420). The cDNA was interpreted as the product of a novel gene, which was assigned the name SLC4A6. Subsequent to this study was a report of a full-length NBCn1 cDNA, called mNBC3, from human skeletal muscle (765). Unfortunately, because of differences between the SBC2/hNBC2 and mNBC3 cDNA sequences,56 mNBC3 was interpreted as the product of a novel gene, which was assigned the name SLC4A7 (765).57 By the time NBC2 and NBC3 had been rationalized as alternative products of the same gene (139, 189), the designation SLC4A7 had already been introduced to the literature. As a consequence, the designation SLC4A6, which had never been mentioned in the literature, was withdrawn. Following the characterization of a rat Slc4a7 product as an electroneutral Na/HCO3 transporter, the gene product was renamed NBCn1 (189). The confusion caused by this changeable nomenclature was fortunately minimal. Few papers refer to NBC2 or SBC2 (344, 426, 427, 457, 485, 814, 989) and fewer still refer to NBCn1, NBC2, SBC2, and NBC3 as though they are the products of distinct genes (344, 426, 457, 485, 530). We further note that NBC2 has been used on one occasion to refer to NBCe1-B (485) and XNBC2 to refer to a Xenopus Slc4a11 product (1102). Due to the withdrawal of the name “NBC2” and because the term “NBC3” is degenerate (i.e., it has been used to refer to both Slc4a7 and Slc4a8 products), we consider NBCn1 as the preferred nomenclature for Slc4a7 products. C) MOLECULAR ACTION OF NBCn1. NBCn1 is an electroneutral Na/HCO3 cotransporter with an associated HCO3⫺-independent conductance (FIGURE 30; 30; Ref. 189), conclusions supported by the following observations.

I) NBCn1 is an electroneutral NCBT with poor DIDSsensitivity. In Xenopus oocytes subjected to a CO2/ HCO3⫺-induced acid load, rat NBCn1 mediates a pHi recovery that is electroneutral, insensitive to 5-(N-ethyl-

56 In addition to numerous artifacts in the hNBC2/SBC clone, the inclusion in mNBC3 versus hNBC2/SBC2 of a different complement of splice cassettes also misled investigators to believe that these two cDNAs were transcribed from different genes. 57 This report was concurrent with a report of a partial product of truly novel gene (Slc4a8), which was also then named NBC3 (35). This product has since been renamed NDCBE.

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III) Urinary system. A) Metabolic acidosis. NBCe2-null mice exhibit a compensated metabolic acidosis that is evidenced by normal blood pH, reduced plasma CO2 and [HCO3⫺], and elevated urinary HCO3⫺ excretion (341). These observations are consistent for a role for NBCe2 in HCO3⫺ reabsorption. However, the NBCe2-null mice also exhibit an increased abundance of pendrin (341), which secretes HCO3⫺ in the collecting duct (reviewed in Ref. 1016). Thus the urinary phenotype might not be solely related to loss of NBCe2 function. Another, untested, possibility is that the acidosis is secondary to disrupted hepatobiliary interactions (e.g., reduced glutamine synthesis).

tines); and 4) the kidney, where NBCn1 promotes NH4⫹ excretion. In keeping with its presence in the eye and ear, mice that lack NBCn1 are both blind and deaf. Multiple studies report genetic linkage between the SLC4A7/NEK10 gene locus and breast cancer in humans. Although the genetic basis underlying the linkage has not been elucidated, the association between pHi regulation and tumor viability is clear.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

NBCn1 Na+

HCO3–

2 Na+ CO32–

Na+ A

Na+

CO32–

Na+ B

NaCO3–

Na+ C

HCO3–

Na+ D

HCO3–

FIGURE 30. Molecular action of NBCn1. Possible molecular mechanisms by which an electroneutral NCBT ⫺ ⫺ could operate with an apparent Na⫹:HCO3 stoichiometry of 1:1. NBCn1 also exhibits a HCO3 -independent conductance that is represented by the red dashed arrow. Note that we have not included any models that are based on CO3/H cotransport, such as those represented for NBCe1 in FIGURE 16.

II) NBCn1 operates independently of Cl⫺. In Xenopus oocytes, the HCO3⫺ efflux mediated by NBCn1, when operating in “reverse” (i.e., initiated by the removal of extracellular Na⫹), does not depend on extracellular Cl⫺ (189). Nor does the HCO3⫺ influx mediated by NBCn1, when operating in the “forward” direction, result in the net efflux of Cl⫺ from the cell (719). Thus NBCn1 is not a Na⫹-driven Cl-HCO3 exchanger. III) NBCn1 has an associated cation leak. After several days incubation, Xenopus oocytes expressing rat NBCn1 are unusually depolarized and loaded with nearly 40 mM [Na⫹]i, more than six times the [Na⫹]i of H2O-injected control oocytes from the same batch (189). Furthermore, NBCn1expressing oocytes, even in the nominal absence of CO2/ HCO3⫺, are substantially hyperpolarized by the removal of extracellular Na⫹, indicating the presence of an associated 58

The first report of NBCn1 expression in oocytes characterized the protein as being DIDS-insensitive, EIPA-sensitive, and capable of substantial Na/OH cotransport or Na-H exchange (765). Subsequent work published with members of the same laboratory reports that HEK-293-expressed NBCn1 does not have these qualities (711). Thus the authors conclude that the EIPA sensitivity and Na/OH cotransport are a feature of NBCn1 expression in oocytes. It is therefore likely that the initial report was confounded by endogenous NHE activity, and possible that none of the reported acid-base transport in fact represented NBCn1. In the meantime, an apical EIPA-sensitive Na-base cotransport activity in ␣-intercalated cells from the rabbit collecting duct (from the inner stripe of the outer medullary) was attributed to NBCn1 in two studies (774, 1083), guided by the original report of EIPA sensitivity of NBCn1 and the apical distribution of NBCn1 suggested by the use of the “anti-NBC3” antibody discussed in Appendix VII. Finally, in aortic smooth muscle of rats (a site of NBCn1 expression), the authors report both an EIPA-sensitive NCBT activity and a distinct SITS-sensitive, and unusually EIPA-sensitive, NDCBE-like activity (592).

Na⫹-conductance pathway (189), that is also a feature of NBCn1 expressed in HEK 293 cells (201). About 50% of the current through the conductive pathway is carried by Na⫹ (189, 201), and Na⫹ conduction is estimated to be responsible for 1/300 of the total Na⫹ movement though the transporter (201). The reversal potential of the conductance varies in a less than Nernstian manner with respect to extracellular [Na⫹], which indicates that the conductance cannot be explained by a simple Na⫹-channel model (201). It is not clear what carries the remainder of the current. Indeed, the Na⫹-independent component of the current remains even in the absence of all extracellular ions except Mg2⫹, Cl⫺, and HEPES, perhaps indicative of an anion-efflux component. The magnitude of the conductance exhibited by membranes of NBCn1-expressing oocytes is not reduced in cells preincubated in Cl⫺-free media, although the Cl⫺ depletion of these cells was not demonstrated (201). The Na⫹-conductive pathway neither depends on nor carries HCO3⫺ and is paradoxically stimulated by DIDS exposure (189). A preliminary report shows that both the HCO3⫺transport and Na⫹-conductive elements of NBCn1 function are upregulated by coexpression of NBCn1 with the NCBT binding partner IRBIT (722), further evidence that the two components may be mediated by the same protein. Finally, a study of NBCe1/ NBCn1 chimeras indicates that the Na⫹ conductance requires elements in the back half of the TMD (i.e., TM6-TM14, inclusive: subdomain 8 in FIGURE 15) of NBCn1 (193). D) THE SLC4A7 GENE.

The human SLC4A7 gene was originally mapped to chromosome 3p22 (766), although more recent genomic assembly suggests that 3p24.1 is a more accurate assignment.59 SLC4A7 occupies at least 27 exons spread over ⬃100 kb (FIGURE 31A). The upstream neighbor of SLC4A7 is the T-box region (TBR) gene EOMES, aka TBR2, which encodes the neuronal transcription factor eomesodermin (495). The SLC4A10 gene, which encodes a second electroneutral Na/HCO3 cotransporter, also has a TBR gene as its

59 A 67-nt sequence with 87% identity to a portion of SLC4A7 (exon 10, encoding sequence in the Nt) is found on chromosome 1p36, 13 kb upstream of the RhD gene. There is currently no evidence to suggest that this sequence is ever transcribed, or is part of an miRNA sequence.

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N-isopropyl)amiloride (EIPA),58 and dependent on both Na⫹ and HCO3⫺, consistent with the activity of an electroneutral NBC (189). Human NBCn1 also mediates a Na⫹-dependent pHi recovery when overexpressed in HEK-293 cells (711). Unusually for an NCBT, NBCn1 cotransport activity is not greatly sensitive to DIDS; in oocytes, 500 ␮M inhibits only 25% of the NBCn1-mediated pHi recovery (189), whereas the Ki for DIDS of the related transporter NBCe1 is ⬃40 ␮M (612).

MARK D. PARKER AND WALTER F. BORON

A

Locus 3p24.1 20 kb

SLC4A7

NEK10

EOMES/TBR2

B

Gene structure

P1

P2

1

2

C

10 kb

3

7 8

26

27

Transcript variation P2 M

3

4

5

6

7

8

9

25

* 27

1

3

4

5

6

7

8

9

25

* 27

1

3

4

5

6

7a

8

9

25

26

* 27

1

3

4

5

6

7

8

9

25

26

* 27

1

3

4

5

6

7

9

25

NBCn1-A

2 M

NBCn1-B M

NBCn1-C M

NBCn1-D M

NBCn1-E

* 27

FIGURE 31. SLC4A7 gene structure and NBCn1 transcript variants. Scale diagrams showing the human SLC4A7 gene locus together with the position of neighboring genes (A), the position of promoters (P1 and P2), and the position of exons within SLC4A7 (B). Transcript variants NBCn1-A to NBCn1-E, which among themselves display the diversity of NBCn1-A to NBCn1-E, are represented, not to scale, as numbered boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature transcript. “//” denotes that all transcripts include exons 9 –25. Exons that include the initiator ATG codon (“M”) and termination codon (“*”) are marked for each transcript. Sequence that is derived from part of a larger exon sequence are labeled with an “a” (e.g., exon 7a is a subdivision of exon 7). Colored exons, or parts of exons, correspond to the protein regions that each encodes, which are identically colored in FIGURE 32. Uncolored exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line are predicted, but not demonstrated, to be included in the mRNA.

upstream neighbor (FIGURE 39A), suggesting a longstanding association between the two gene families, and one that predates the duplication of this gene region. E) STRUCTURAL FEATURES AND VARIANTS OF NBCn1. Slc4a7 products that encode full-length transporters are currently named NBCn1-A through -L and are the most diverse in terms of number of reported and predicted variant transcripts. Here we describe the nature of the variant features (i.e., alternative promoters, splice cassettes), followed by a description of NBCn1-A through -L. A diagrammatic representation of the sources of transcript variation is provided in FIGURE 31C. A representation of each NBCn1 protein variant is provided in FIGURE 32.

I) Sources of variation in coding sequence among NBCn1 variants. Alternative promoter choice and Nt (“MERF”- versus “MEAD”-). Mammalian NBCn1 transcription can initiate

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at either exon 1 or exon 2, presumably dictated by a choice of alternative promoter regions (FIGURE 31B). The result is variant protein products whose Nt begins either with an 11amino acid or a 16-amino acid protein cassette. In humans, exon 2, which encodes the 11-amino sequence (beginning with the sequence MERF), is located 4 kb upstream of exon 3 in the SLC4A7 gene, whereas exon 1, which encodes the 16-amino acid sequence (beginning with the sequence MEAD), is 32 kb upstream of exon 3. The consequences of this promoter choice for NBCn1 function and/or regulation are unclear. The full-length NBCn1 protein variants that begin with “MERF” are NBCn1-A, -F, -J, -K, and -L, whereas the variants that begin with “MEAD” are NBCn1-B, -C, -D, -E, G, -H, and -I. A) Variation in the length of “MEAD”-encoding exon 1 (NBCn1-X=). In some variants, the 3= boundary of exon 1 is extended by the use of an alternative splice site such that the

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P1

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

III 6–9

10–14

124

1,214

124

1,219

124

PDZ

NBCn1-B

1–5

PDZ

11

Ct

PDZ

NBCn1-A

TMD

C as se tte

100 aa

C as se tte I C as se tte II

Nt

1,242

13

16 NBCn1-C

36 124

NBCn1-E NBCn1-F NBCn1-G NBCn1-H

124

NBCn1-I NBCn1-J NBCn1-K

124

NBCn1-L

1,255 1,095 1,090 1,131 1,206 1,118 1,126 1,201 1,113

FIGURE 32. NBCn1 protein variants. Scale diagram of protein variants that are encoded by the transcripts represented in FIGURE 31C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino acids and colored to denote their genetic origin as shown in FIGURE 31C. All NBCn1 variants are presumed to include an autoinhibitory domain and IRBIT-binding determinants in their Nt. All NBCn1 variants terminate with a PDZ binding motif. A color-matched protein sequence alignment of the variants is provided in Appendix V.

16-amino acid sequence is lengthened at its carboxy-terminal end by the 4-amino acid sequence “VTSR”. The terminology for such variants has not been settled. Some have been designated either with a prime (e.g., NBCn1-D= is identical to NBCn1-D except for the inclusion of “VTSR”; Appendix IV and Appendix V) and others with a lowercase “a” and “b” (e.g., NBCn1-Hb is identical to NBC1-Ha except for the inclusion of “VTSR”). The consequences of the inclusion of “VTSR” are unknown. B) Cassette I (aka “cassette A”). A 13-amino acid “cassette I” (originally termed “cassette A” in Ref. 189)60 is encoded by a 3= extension of exon 7 (FIGURE 31C). The protein

60 Analysis of the Slc4a7 gene structure leads us to deduce that cassette I encompasses the 13-amino acid sequence “GKKHSD PHLLERN” and not, as originally deduced in Ref. 189, the 14-amino acid sequence “GKKHSDPHLLERNG.”

sequence encoded by cassette I is located in the Nt loop subdomain (FIGURE 15) and is absent from some variants of NBCn1 (FIGURE 32). The consequences of this splice for NBCn1 function and/or regulation are unclear. Several studies have used RT-PCR to address the spatial distribution of splice cassette I, as well as II and III (discussed below). The most exhaustive to date is presented in Reference 213. In preparations from various organs/tissues of adult mice, NBCn1 splicing generally does not appear to favor the omission or inclusion of cassette I (55, 213). However, a preference for cassette I inclusion seems to exist in kidney cortex, submandibular gland, parotid gland, and liver. Conversely, a preference for cassette I exclusion appears to exist in the lung.⬎ The full-length NBCn1 protein variants that include cassette I are NBCn1-A, -B, -D, -E, -F, -G, and -J. Variants lacking cassette I are NBCn1-C, -H, -I, -K, and -L. Among

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PDZ PDZ PDZ PDZ PDZ PDZ PDZ PDZ PDZ

NBCn1-D

MARK D. PARKER AND WALTER F. BORON those variants lacking cassette I, at least NBCn1-H is functional.

Comparisons of Xenopus-oocyte– expressed NBCn1 variants, with or without cassette II (specifically NBCn1-B versus NBCn1-E), indicate that NBCn1 ⫹ cassette II accumulates in the plasma membrane more slowly than NBCn1 ⫺ cassette II (1078). However, after 72 h of expression, both variants accumulate to a similar extent in the oocyte membrane, and both mediate a Na⫹ conductance of equivalent magnitude (1078). Some differences are evident between the two variants at low [Na⫹]o: 1) NBCn1 ⫹ cassette II appears to have a lower Na⫹ affinity, and 2) the variants exhibit differences in the appearance of a conductive leak of an as-yet-unidentified nature (1078). The reduced functional expression of NBCn1 ⫹ cassette II is also indirectly evident in an opossum kidney cell line, inasmuch as the endogenous Na pump in these cells is less active in cells coexpressing NBCn1 ⫹ cassette II than in cells coexpressing NBCn1 ⫺ cassette II, presumably because the less active variant of NBCn1 imposes a lesser Na⫹ load on the cells (1078). Thus cassette II appears to be inhibitory in at least certain contexts, perhaps because it serves as a binding site for calcineurin or other proteins with an inhibitory effect. In mice (55, 213) and in rats (200), most NBCn1 transcripts lack cassette II, as assessed by PCR across a region including cassette II. However, the inclusion of cassette II appears to

61 “Exon 7” is a misnomer. Although cassette II is encoded by the seventh exon of individual NBCn1 transcripts, cassette II is encoded by exon 8 of the Slc4a7 gene (see FIGURE 31C).

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The full-length NBCn1 protein variants that include cassette II are NBCn1-A, -B, -C, -D, -H, and -K. Variants lacking cassette II are NBCn1-E, -F, -G, -I, -J, and -L. Variants that include both cassettes I and II are NBCn1-A, -B, and -D. The only two variants that lack both cassettes I and II are NBCn1-I and -L. D) Cassette III (aka “cassette B”). The final published source of NBCn1 variation lies in the Ct of the protein (FIGURE 32). NBCn1 variants are unique among NCBTs in that the sequence of the extreme Ct is invariant: all variants terminate with the PDZ-binding domain ligand -ETSL. Instead, Ct variation among NBCn1 forms is achieved by the optional inclusion of a 36-amino acid “cassette III” (originally termed “cassette B” in Ref. 189). As far as other NCBTs are concerned, cassette III–like sequence is also present in the Ct of NDCBE-A and NDCBE-C, and inclusion of cassette III–like sequence is obligatory in the Ct of all NBCn2 variants (see extended domain alignment in Appendix V). Experiments on oocytes expressing NBCn1 variants indicate that cassette III stimulates overall functional expression in the absence (but not in the presence) of cassette II (compare NBCn1-G versus -E and -D versus -B). In preparations from various organs/tissues of adult mice, NBCn1 splicing generally does not appear to favor the omission or inclusion of cassette III (55, 213). However, in rodents, a preference for cassette III inclusion seems to exist in kidney cortex (213); renal mTAL (694); submandibular gland (213), specifically the acini, see Reference 615; sublingual gland (213); pylorus (213); colon (213); pancreas (213); lung (213); cerebrum (213) and certain other areas of the brain (755); and epididymis (213). On the other hand, a preference for cassette III exclusion appears to exist in cardiac ventricles (213) and in the ducts of submandibular glands (615). The full-length NBCn1 protein variants that include cassette III are NBCn1-C, -D, -G, I-, -J, and -L. Variants lacking cassette III are NBCn1-A, -B, -E, -F, -H, and -K. Of the 12 confirmed transcripts, only NBCn1-D includes all three cassettes I, II, and III. II) Cloned NBCn1 variants that are demonstrated or likely to exhibit NCBT activity. The known NBCn1 variants are listed below along with their features and their demonstrated locations. Information about clones that have not yet been reported in a full manuscript is derived from their GenBank entries (accession numbers are provided in Appendix IV).

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C) Cassette II (aka “exon 7”).61 Abutting cassette I, and also within the Nt loop subdomain (FIGURE 15), is the 124amino acid cassette II (123 amino acids in rodents due to the lack of an Ala residue close to the Ct end of the cassette), encompassing the entirety of exon-8 – encoded sequence (FIGURES 31C AND 32). Cassette II sequence is unlike that encoded by any other mammalian gene, but contains a number of consensus PKA and PKC phosphorylation sites and, as an extension of the Nt loop subdomain, is likely accessible to a number of cytosolic binding partners. A preliminary report demonstrates that an isolated cassette II is able to interact with calcineurin-A␤ in vitro (715). Indeed, cassette II includes a motif “PTVVIH” that is similar to a consensus calcineurin-binding motif (45). Moreover, perturbation of this sequence disrupts the in vitro interaction between isolated cassette II and calcineurin (715). The physiological relevance of this interaction remains untested, although perhaps pertinent is the observation that NBCn1 protein abundance is decreased in the renal medulla of rats treated with the calcineurin inhibitor FK506 (655).

be favored in rodent aorta (55, 200), rat heart (fetal ⬎ adult; interventricular septum ⬎ ventricles ⬎ auricles ⬎ atria ⬎ AV node; Ref. 200), human heart and muscle (200, 765), and in fetal rat hippocampal neurons (201).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

A) NBCn1-A (NCBT activity demonstrated). NBCn1-A is the archetypal human NBCn1 clone (765). NBCn1-A initiates with the “MERF” Nt, includes cassette I and II, but omits cassette III. A full-length NBCn1-A clone has been isolated from a human skeletal muscle cDNA preparation (765). B) NBCn1-B (NCBT activity demonstrated). NBCn1-B is the archetypal rat NBCn1 clone (189). NBCn1-B initiates with the “MEAD” Nt, includes cassette I and II, but omits cassette III. A full-length NBCn1-B clone has been isolated from rat aorta (189) and embryonic rat hippocampal neuron cDNA preparations (201).

D) NBCn1-D and D= (NCBT activity demonstrated). NBCn1-D initiates with the “MEAD” Nt sequence, and includes cassettes I, II, and III. NBCn1-D= is the most complete “MEAD”-initiated NBCn1 clone as it initiates with the extended 20-amino acid “MEAD” Nt sequence and also includes all three splice cassettes. Full-length NBCn1-D has been isolated from rat aorta cDNA preparations (189). Full-length NBCn1-D= has been isolated from human liver cDNA preparations. E) NBCn1-E (NCBT activity demonstrated). NBCn1-E initiates with the “MEAD” Nt, includes cassette I, but omits cassettes II and III. Full-length NBCn1-E clones have been isolated from human skeletal muscle, adult-rat hippocampal neurons (201), and mouse reproductive tract cDNA preparations. F) NBCn1-F (NCBT activity untested). NBCn1-F initiates with the “MERF” Nt, includes cassette I, but omits cassettes II and III. Full-length NBCn1-F clones have been isolated from human kidney cDNA preparations. G) NBCn1-G (NCBT activity demonstrated). NBCn1-G initiates with the “MEAD” Nt, includes cassettes I and III, but omits cassette II. Full-length NBCn1-G clones have been isolated from human skeletal muscle cDNA preparations. H) NBCn1-H and H= (NCBT activity demonstrated). NBCn1-H initiates with the “MEAD” Nt, includes cassette II, but omits cassettes I and III. NBCn1-H= is the same but initiates with the extended 20-amino acid “MEAD” Nt sequence. Full-length NBCn1-H and -H= clones have both

I) NBCn1-I (NCBT activity untested). NBCn1-I initiates with the “MEAD” Nt, includes cassette III, but omits cassettes I and II. Full-length NBCn1-I clones have been isolated from mouse reproductive tract cDNA preparations. J) NBCn1-J (NCBT activity untested). NBCn1-J initiates with the “MERF” Nt, includes cassettes I and III, but omits cassette II. Full-length NBCn1-I clones have been isolated from mouse ovary and testis cDNA preparations. K) NBCn1-K (NCBT activity untested). NBCn1-K initiates with the “MERF” Nt, includes cassette II, but omits cassettes I and III. Full-length NBCn1-I clones have been isolated from mouse skeletal muscle cDNA preparations. L) NBCn1-L (NCBT activity untested). NBCn1-L initiates with the “MEAD” Nt, includes cassette III, but omits cassettes I and II. Full-length NBCn1-L clones have been isolated from mouse reproductive tract cDNA preparations. III) Predicted NBCn1 variants. The choice of three alternative first exons and the omission or inclusion of any of the three splice cassettes I, II, or III could, together, produce as many as 24 variants, although presently only 15 of the 24 have been cloned as full-length cDNAs.62 No pattern of association between promoter/cassette usages has emerged that suggests any of the “missing” combinations63 are unfavored; thus it is likely that their existence will be documented in due course. IV) Other NBCn1 variants. A) Unusual variants that represent only the isolated Nt. Six unusual cDNA species from brain, heart, and skeletal muscle cDNA are identical to full-length NBCn1 transcripts except for the omission of exon 13 (GenBank DNA accessions nos. FJ178574, FJ178575, FJ178576, GU354307, GU354309, and GU354310).64 If translated, each of these cDNAs is predicted to produce a soluble protein that would include 62 In a personal communication, Drs. Liming Chen and Ying Liu report to us the existence of a new promoter and a new cassette that could increase the number of possible variants to 64. 63 Considering only “MEAD” versus “MERF” and the three cassettes, the “missing” combinations are MEAD/-I/-II/-III, MERF/⫹I/ ⫹II/⫹III, MERF/-I/-II/⫹III, MERF/-I/⫹II/⫹III, and MERF/-I/-II/-III. 64 If these clones included exon 13, FJ178574 would encode NBCn1-H, FJ178575 would encode NBCn1-G, FJ178576 would encode NBCn1-E, GU354307 would encode NBCn1-C, GU354309 would encode NBCn1-E=, and GU354310 would encode NBCn1-G=. Because some of these full-length NBCn1 clones differ only in the protein sequence of their Ct, the accession pair FJ178574/ GU354307 is predicted to encode identical isolated-Nt polypeptides as are the pairs FJ178575/FJ178576 and GU354309/ GU354310.

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C) NBCn1-C and C= (NCBT activities untested). NBCn1-C initiates with the “MEAD” Nt sequence, includes cassette II, but omits cassettes I and III. NBCn1-C= is the same but initiates with the extended 20-amino acid “MEAD” Nt sequence. A full-length NBCn1-C clone has been isolated from rat aorta cDNA preparations (189). A full-length NBCn1-C= clone has been isolated from human skeletal muscle cDNA preparations.

been isolated from human skeletal muscle cDNA preparations.

MARK D. PARKER AND WALTER F. BORON almost the entire “MEAD” Nt (i.e., the sequence encoded by exons 1 and 3–12, per normal) plus two residues “VQ” followed by a termination codon (encoded by an out-of-frame exon 14). The protein would be truncated at a point 25 amino acids upstream of TM1, and thus would precisely terminate in the region that, in AE1, is predicted to be an unstructured linker that join the Nt to the TMD. The physiological relevance of these clones remains obscure, and the cognate protein has yet to be identified. It is possible that the premature termination codons included in these unusual mRNAs would make them targets for nonsense-mediated decay (170). These clones are reminiscent of isolated Nt variants of NDCBE and NBCn2.

F) DISTRIBUTION OF NBCn1. The distribution of NBCn1 is broad, as summarized in TABLE 5. Its location in specific organ systems is discussed below. Note that the tissue distributions of the alternate promoters and three splice cassettes were discussed above in section VB5 on p. 882.

I) Central nervous system. A) Brain. At the level of subregions of the central nervous system, NBCn1 transcripts (755) and protein (174, 709) are widespread, being present in the cerebral cortex, hippocampus, subcortex, cerebellum, and olfactory bulb of rodents. NBCn1 immunoreactivity in wild-type rats and ␤-galactosidase staining of heterozygous mice with a lacZ insertion in the Slc4a7 gene shows that the highest level of NBCn1 promoter activity, at the tissue level, is in the pyramidal cell layers of the hippocampus, appearing equally robust in the CA1, CA2, and CA3 regions, and in the granule cells layer of the dentate gyrus (91, 709). In these “lacZ” mice, NBCn1 promoter activity is also evident in certain regions of the cortex and the dentate nucleus of the cerebellum (91). At the cellular level, NBCn1 expression is detected in hippocampal neurons of embryonic rats (201) and adult mice (91). In primary cultures of hippocampal neurons from embryonic rats, most cells, both GABAergic and non-GABAergic, express NBCn1 transcripts (201). NBCn1-like activity is detected in locus coeruleus neurons (479), although the presence of NBCn1 transcripts and protein in these cells has yet to be demonstrated. At the subcellular level in embryonic rat neurons, NBCn1 protein is detected in the plasma membrane of the cell soma as well as in the dendrites, with a punctuate distribution that is consistent with a presence in the dendritic spines (201, 709). B) Choroid plexus. NBCn1 protein has a basolateral presence (see FIGURE 28) in the choroid plexus of rats (213, 709, 755), some strains of mice (755), and humans (756). NBCn1 also has a small apical presence in the choroid plexus of human ventricle IV (756) and is predominantly apically expressed in some strains of mice (470, 755).

NBCn1 distribution has been the focus of much investigation, revealing some discrepancies between studies. That is to say, certain NBCn1-directed polyclonal antibodies suggest a different transporter distribution than others. Some studies report that the localization of NBCn1 in a tissue may vary among species (485) and even among mouse strains (216). Data concerning NBCn1 protein distribution come from the use of the three types of antibodies, those that are generated against: 1) epitopes in the Ct of rat NBCn1 (1014), 2) epitopes in the Ct of human NBCn1 (“anti-NBC3”; Ref. 774), and 3) an epitope in the Nt of rat NBCn1 (213). The studies using antibodies 1 and 3 reinforce each other and are confirmed by ␤-galactosidase staining in mice heterozygous for the insertion of lacZ into the Slc4a7 gene (91). It is therefore the results of these studies that are cited here for NBCn1 protein localization.

II) Sensory organs. A) Eye. NBCn1 promoter activity is evident in the photoreceptor and ganglion cells of mouse retina (91) and NBCn1 transcripts have been detected in Northern blots of rabbit eyes (814). Microarray analysis demonstrates that cultured mouse keratocytes express NBCn1 transcripts (see Gene Expression Omnibus database entry GDS85765 that accompanies Ref. 162). See Appendix VII for subretinal distribution based on the “antiNBC3” antibody.

The distribution of NBCn1 as reported by antibody 2, the “anti-NBC3” antibody, is unusual in many respects (dis-

65 Gene Expression Omnibus Entry at (http://www.ncbi.nlm.nih. gov/sites/GDSbrowser?acc⫽GDS857).

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B) Ear. See Appendix VII for distribution of NBCn1 within the inner ear according to the “anti-NBC3” antibody.

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B) SBC2/hNBC2 (probable cloning artifact). The SBC2/ hNBC2 cDNA sequence (GenBank protein accession no. BAA25898) was the first reported SLC4A7 product (420). It differs from subsequently reported SLC4A7 products in three respects: 1) hNBC2 lacks exons 0 – 4 of verified SLC4A7 transcripts. 2) The 5=-UTR and the initial portion of the purported open reading frame of hNBC2 is derived from the exons 5 and 6 of the SLC4A7 gene, but this sequence has an inverted orientation with respect to the remainder of the transcript. 3) The 3= end of the hNBC2 transcript is not predicted by the genomic sequence, presumably due to low fidelity of the amplified cDNA: because of this the ORF contains three missense mutations and reaches a premature stop, 18 amino acid short of the Ct end of verified Slc4a7 products.

cussed in Ref. 334) and is considered separately in Appendix VII (see section V).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

III) Peripheral nervous system. A) Trigeminal ganglion. In rats, NBCn1 transcripts are detected by RT-PCR in trigeminal ganglion preparations (408).

B) Osteoblasts. A proteomic study reveals NBCn1 protein to be present in the hydroxyapatite-releasing microvesicles that bud from the apical membranes of osteoblasts (969).

IV) Respiratory system. A) Trachea and lung. NBCn1 transcripts and protein are present in preparations of rat lung (189, 213), and NBCn1 transcripts are present in the Calu-3 airway cell line (see “NBC2” in Ref. 515). NBCn1 promoter activity is evident in nonvascular smooth muscle cells of mouse trachea (91).

C) Osteoclasts. Osteoclasts express NBCn1 protein (112, 797), specifically in the ruffled membrane that faces the bone resorption lacuna (see cartoon in FIGURE 33 and ref. 797).67

B) Vasculature. As noted in the previous paragraph, NBCn1 is present in cardiac endothelial cells. In vascular smooth muscle, NBCn1 cDNA has been amplified from pulmonary artery and aorta (189), and NBCn1 protein is detected in the portal vein, and in the hepatic, mesenteric, and intrarenal cortical arteries of rats. NBCn1 protein is also present in rat skeletal muscle vasculature. In the arteries, it is the endothelial cells and the smooth muscle cells of the tunica media that are NBCn1 positive (213). LacZ/␤galactosidase staining provides evidence for NBCn1 promoter activity in mouse cerebral arteries and veins, mesenteric arteries, and renal arteries of mice (91). In the vascular smooth muscle of a mouse mesenteric small artery, immunogold staining detects NBCn1 expression in the sarcolemmal membrane (90). VI) Musculoskeletal system. A) Skeletal muscle. The archetypal human NBCn1 clone was amplified from skeletal muscle cDNA. Within skeletal muscle of rats, NBCn1 protein localizes to vasculature as well as to the vicinity of neuromuscular junctions. However, NBCn1 does not colocalize with ␣-bungarotoxin, suggesting that NBCn1 is present in motor neuron terminals or sarcolemmal areas that lack the nicotinic acetylcholine receptor (213).

66 The antibody in this study detected only NH2 termini beginning with “MEAD” (see green cassette in FIGURE 32).

B) Salivary gland. In human, but not rat, parotid and submandibular salivary glands, NBCn1 protein is enriched in the basolateral membranes of the striated duct cell (FIGURE 21B). However, NBCn1 is not detected in acinar cells (334), which are a site of basolateral NBCe1 expression (FIGURE 21A). NBCn1 is expressed in the basolateral membranes of an immortalized cell line from rat parotid acini (740). The evidence for the presence of NBCn1 in the apical membrane of salivary gland duct cells is indirect, being based on Co-IP of NBCn1 and CFTR in isolated tissues, rather than immunohistochemical data (711). See Appendix VII for distribution of NBCn1 within the salivary glands according to the “anti-NBC3” antibody. C) Stomach. Results of qPCR show that rabbit NBCn1 transcripts are expressed in gastric mucosa, most prominently in the chief cells and mucous cells, with lesser expression in parietal cells (814). LacZ/␤-galactosidase staining is negative in mouse gastric mucosa (91). VIII) Lower digestive system. A) Intestines. In rabbits, NBCn1 transcripts are more abundant in the duodenal and ileal mucosa than in either gastric or colonic mucosa (427). In mice, NBCn1 transcripts are detected in duodenal and colonic epithelia (55, 180, 213). In mice, anti-NBCn1 antibodies localize NBCn1 protein to the basolateral membrane of the enterocytes of duodenal villi (see FIGURE 22 as well as Refs. 180, 213, and 753). The presence of NBCn1 in the enterocytes of mouse duodenal villi, but not of crypts, is confirmed by lacZ/␤-galactosidase staining of NBCn1/lacZ transgenic mice (91). In the colons of NBCn1/lacZ mice, ␤-galactosidase staining reveals the presence of Slc4a7 products in villar epithelial cells, but not in crypt epithelial cells (91). In colonic crypts, qPCR indicates a low level of NBCn1 transcript expression that is swamped by an ⬃80fold greater abundance of NBCe1-B transcripts (1087).

67 The osteoclast membrane has a ruffled border (facing the resorption lacuna) and a free surface (not facing the lacuna). Although these domains are sometimes considered equivalent to the apical and basolateral membranes of epithelia, the free surface is composed of subdomains that contain both apical and basolateral markers of classic epithelia, whereas the ruffled border could be considered as a “giant extracellular lysosome” (662, 991).

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V) Circulatory system. A) Heart. In human heart, NBCn1 cDNAs have been amplified from tissue dissected from aorta, apex, atria, auricles, ventricles, interventricular septum, and atrioventricular node (200). In rat heart, NBCn1 protein66 is found in myocardial capillaries, the endothelium and vasa vasorum of aorta, and in the endothelia of atria and ventricles. In ventricular endothelia, immunogold staining reveals NBCn1 protein in both the luminal and abluminal (i.e., basal) membranes (213). ␤-Galactosidase staining of mice with a lacZ insertion in Slc4a7 provides evidence for NBCn1 promoter activity in the aorta and in cardiac myocytes of the atria, but not the ventricles (91). A preliminary study reports a diffuse NBCn1 immunoreactivity in rat ventricular myocytes (311).

VII) Upper digestive system. A) Enamel organ. NBCn1 immunoreactivity is reported in the papillary cell layer (456) of the enamel organ (FIGURE 20).

MARK D. PARKER AND WALTER F. BORON

HCO3–

Ca2+ Osteoclast NCX

AE2 3 Na+ Cl–

HCO3–

CO2 CAII

H+

H2O

H+ Na+

++

NHE

cAMP Ca2+

HCO3– Na+ H-pump

NCX

NBCn1

Sealing zone

Bone matrix

3 Na+

H+ CaCO3

Resorption lacuna

FIGURE 33. Role of NBCn1 in osteoclasts. Intracellular carbonic anhydrase generates H⫹ that are secreted by the H-pump into the resorption lacuna to dissolve bone minerals. H⫹ secretion is supported by AE2 in the ⫺ is absorbed across the lacunar membrane by NBCn1 and across contra-lacunar membrane. Liberated HCO3 the contra-lacunar membrane by AE2. Liberated Ca2⫹ is absorbed by the combined actions of NCX and TRPV5 channels (580, 994).

LacZ/␤-galactosidase staining of NBCn1/lacZ mice reveals that some of the NBCn1 transcripts detected by qPCR in the duodenum and colon, and the majority of the NBCn1 transcripts detected in the jejunum and ileum (180), represent NBCn1 expression in the nonvascular smooth muscle cell layers, rather than the epithelium (91). B) Liver. Slc4a7 products have been amplified from cDNA preparations of rat and mouse liver (189, 213). See Appendix VII for hepatic distribution of NBCn1 according to the “anti-NBC3” antibody. C) Pancreas. The detection of NBCn1 transcripts in preparation of mouse pancreas are reported as unpublished data by Xuo and Muallem (711). Furthermore, ESTs appear to be common in preparations from mouse pancreas (Appendix VI). See Appendix VII for pancreatic distribution of NBCn1 according to the “anti-NBC3” antibody. IX) Lymphatic and immune systems. A) Spleen and macrophages. Slc4a7 products have been amplified from cDNA preparations of macrophages (630) and spleen (189, 213).

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X) Endocrine system. A) Thyroid. According to an NCBIcurated database of ESTs, the human thyroid gland is a site of NBCn1 transcription (Appendix VI). XI) Urinary system. A) Kidney. NBCn1 transcripts are amplified from cortical preparations from rabbits (427), as well as the inner stripe of the outer medulla (754), IMCD (986), and mTAL (694) preparations from rats. AntiNBCn1 antibodies detect an 180-kDa protein in western blots of rat preparations of the inner medulla as well as the inner and outer stripes of the outer medulla (213, 1014). An anti-NBCn1-Nt antibody, but not an anti-NBCn1-Ct antibody, detects NBCn1 protein in the renal cortex (213), where NBCn1 expression appears to be less than in the medulla (213). LacZ/␤-galactosidase staining indicates that NBCn1 expression in mouse cortex may predominantly represent vascular expression in the afferent arterioles and renal corpuscles (91). No evidence of NBCn1 promoter activity is detected in cortical collecting ducts (CCDs, Ref. 91). At the cellular level, anti-NBCn1 antibodies detect a basolaterally located protein in mTAL epithelial cells (334, 431, 491, 530, 754, 797, 1014, 1026), intercalated cells in the inner stripe of the outer medulla (1014), ␣-intercalated cells

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TRPV5

Ca2+

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

in the IMCD (694, 1014), and renal papilla epithelial cells (754) of rats. Only an anti-NBCn1-Nt antibody detects NBCn1 protein at the basolateral membrane of a subset of outer medullary collecting ducts (OMCD) intercalated cells (213). This presence of NBCn1 in rat mTAL, IMCD, and OMCD matches the pattern of Slc4a7 promoter activity disclosed by lacZ/␤-galactosidase staining (91). In these mice, renal Slc4a7 promoter activity is particularly robust in the epithelium lining the renal pelvis. See Appendix VII for renal distribution of NBCn1 according to the “anti-NBC3” antibody. B) Bladder. Slc4a7 promoter activity has been detected in nonvascular smooth muscle cells from mouse bladder (91).

See Appendix VII for distribution of NBCn1 in the epididymis according to the “anti-NBC3” antibody. B) Female. NBCn1 transcripts have been detected by RTPCR of mouse ovary, uterus, and vagina (599), and ESTs are abundant in mouse mammary gland preparations (Appendix VI). NBCn1 protein is present in the lobular acini of the human breast (182). Slc4a7 promoter activity has been detected in the myometrium of the uterus of mice (91). G) PHYSIOLOGICAL ROLES OF NBCn1.

We have seen that NBCn1 has a broad distribution throughout the body and likely supports HCO3⫺ secretion across a number of epithelia and contributes to pHi regulation in all of the cell types in which it is located. Further physiological roles are suggested by characteristics exhibited by NBCn1-null mice, although primary versus secondary effects of NBCn1 loss have yet to be distinguished. I) General. A) pHi regulation. DIDS-insensitive (or poorly DIDS-sensitive) Na/HCO3 cotransport, a strong indicator of NBCn1 activity, contributes to pHi regulation in many tissues, including the choroid plexus (113), duodenum (427), renal mTAL (530, 694) and IMCD (754), as well as ureter (13). NBCn1 is also strongly implicated as a contributor to pHi regulation of mouse vascular smooth muscle cells, although, as discussed below, overall Na/HCO3 transport in these cells is partly sensitive to DIDS (90). As discussed in footnote 54, NBCn1 is unlikely to be responsible for the DIDS-insensitive, EIPA-sensitive Na/base transport that has been detected at the apical membrane of OMCD ␣ intercalated cells.

B) Potential contribution to CSF secretion. In choroid plexus epithelia (FIGURE 28), the basolateral presence of NBCn1 protein (213, 755, 756) parallels the distribution of NBCn2. In light of the prominent role played by NBCn2 in CSF secretion (429), it has been suggested that the role played by NBCn1 in CSF formation may be less significant. Indeed, in some strains of mice, NBCn1 in the CPE is predominantly at the apical membrane (216, 470), where the protein would not be in a position to contribute to CSF secretion. Moreover, in NBCn2 and NBCe2 knockout mice, endogenous NBCn1 does not compensate for defective CSF secretion (216, 470). III) Peripheral nervous system. A) Potential contribution to neuronal excitability. Although NBCn1 transcripts are present in neurons cultured from trigeminal ganglions of rats, the NCBT activity in these cells is fully blocked by DIDS, an observation that is inconsistent with the relative DIDS insensitivity of NBCn1 in oocytes (see Ref. 408). IV) Circulatory system. A) Tone and contractility of vascular smooth muscle. In the vascular smooth muscle cells of mice, Na⫹-dependent HCO3⫺ transport makes a major contribution to the recovery of pHi from an acid-load (90). The presence of CO2/HCO3⫺ in the extracellular fluid contributes to enhanced myogenic tone and the ability to maintain contractile ability during sustained agonist exposure, presumably due to transporter-mediated HCO3⫺ uptake. Two pieces of data speak to the importance of NBCn1 in mediating this HCO3⫺ uptake. 1) At the transcript level, NBCn1 is the only Na⫹-dependent Slc4 family member detectable in mesenteric, coronary, and cerebral arteries, and 2) siRNA-directed knockdown of NBCn1 (to ⬃50% normal levels) reduces both the steady-state pHi of these cells and the rate at which their pHi recovers from an acidload (90). One usual aspect of these studies is that the pHi recovery in mesenteric arteries is unusually DIDS sensitive (90) whereas, as mentioned above, NBCn1 activity is relatively DIDS insensitive in other tissues and heterologous expression systems. V) Musculoskeletal system. A) Osteoclast survival and function. Treatment of osteoclasts with CSF-1 results in an increase in pHi via a mechanism that depends on Na⫹ and HCO3⫺, but that is not sensitive to DIDS or EIPA—the hallmarks of NBCn1 activity. Because treatment of osteoclasts with CO2/HCO3⫺ reduces apoptosis in osteoclasts, an effect further promoted by the addition of CSF-1, it has been proposed that NBCn1, by raising pHi and/or [HCO3⫺]i, inhibits caspase activity and thereby promotes osteoclast sur-

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XII) Reproductive system. A) Male. NBCn1 transcripts have been detected by RT-PCR of mouse testis, epididymis, and vas deferens (599) as well as by Northern blot of human testis (420). NBCn1 protein (213) has been detected in preparations of rat epididymis (213).

II) Central nervous system. A) Potential contribution to neuronal excitability. Being widely expressed in neurons throughout the brain, NBCn1 likely contributes towards control of neuronal excitability, as has been demonstrated for other NCBTs, such as NBCe1 (FIGURE 24).

MARK D. PARKER AND WALTER F. BORON vival (112). More recently, NBCn1 has been suggested to play a direct role in reabsorbing the HCO3⫺ liberated from the hydroxyapatite matrix during bone remodeling (797). This observation is supported by the presence of NBCn1 protein in the ruffled membrane that faces the resorption space (lacuna, see FIGURE 33) and the decreased bone absorptive capabilities of osteoclasts when NBCn1 abundance is reduced in these cells by shRNA (797).

that maintains sperm in a quiescent state (771). On the other hand, as discussed above and in Appendix VII, the anti-NBC3 antibody has not been a reliable tool. Independent verification of this apical polarity of NBCn1 distribution is presently lacking. If NBCn1 were instead basolaterally disposed, it could contribute towards HCO3⫺ secretion, and thence fertility, as proposed for NBCe1 (p. 73). H) CAUSES OF NBCn1 UPREGULATION. NBCn1 transcript and pro-

VII) Lower digestive system. A) Transepithelial HCO3⫺ secretion in intestines. NBCn1 is present in the basolateral membranes of epithelia in the lower digestive system (FIGURE 22), enabling the transporter to contribute to the basolateral step of transepithelial HCO3⫺ secretion into the gut lumen and thereby protect the mucosa from gastric acid. The importance of NBCn1 for this process is demonstrated by a substantial reduction in the basal and forskolin-stimulated rates of HCO3⫺ secretion by the duodena of NBCn1null mice (180). VIII) Urinary system. A) Enhancement of renal NH4⫹ excretion. The renal medullary thick ascending limb is a major site of NH4⫹ reabsorption, which occurs as NH4⫹ enters the cell across the apical membrane via NKCC2 and the renal outer medullary K⫹ channel (ROMK) and then sheds a proton—thereby acidifying the cell—to form NH3 (FIGURE 34). This NH3 exits across the basolateral membrane and then enters the medullary collecting duct, where it is trapped as NH4⫹ which appears in the urine and thereby plays a major role in urinary acid secretion (316). One would expect that NBCn1, present at the basolateral membrane of mTAL cells, would tend to neutralize pHi during NH4⫹ reabsorption. Indeed, NBCn1 protein is upregulated during metabolic acidosis and downregulated during metabolic alkalosis. Furthermore, the influx of ammonium and methylammonium in NBCn1-expressing Xenopus oocytes is stimulated in the joint presence of Na⫹ and HCO3⫺ (556, 557). However, in the absence of an acid load, NBCn1-knockout mice lack an obvious renal phenotype (93). IX) Reproductive system. A) Possible role in HCO3⫺ reabsorption and/or secretion in the epididymis. An apical distribution of NBCn1 protein in certain cells along the rat epididymis is indicated by the use of the “anti-NBC3” antibody discussed in Appendix VII. At the apical membrane, NBCn1 would be positioned to reabsorb HCO3⫺ from the epididymal fluid, contributing to the luminal acidification

890

tein abundance are typically increased by maneuvers that elicit an acidosis, reflecting a general pattern of increased abundance of acid extruders (e.g., NBCe1 and NHE1) and decreased abundance of acid loaders (e.g., AE3) under these conditions. I) Central nervous system. A) Increased transcript and protein abundance in brain in response to acidosis. In primary cultures of rat hippocampal neurons, lowering extracellular pH below 6.8, a maneuver that presumably lowers pHi to some extent, results in an increase in NBCn1 protein levels (202). The abundance of NBCn1 transcripts and protein in the brain is increased in a rat model of chronic metabolic acidosis (709). B) Increased protein abundance in response to hypercapnia. Chronic hypercapnia generally increases NBCn1 protein abundance in the neonatal, but not adult, mouse cerebral cortex (463), which may help to counter the acidifying effects of hypercapnia. II) Circulatory system. A) Increased transcript abundance and transporter activity in heart during pressure-overload hypertrophy. Hypertrophy of ventricles in rats with constricted aortas is accompanied by an increase in ventricular NBCe1 and NBCn1 transcript abundance (1071) and an increase in HCO3⫺-dependent acid extrusion in myocytes isolated from the hypertrophic ventricles. The presence of NBCn1 protein has yet to be demonstrated in ventricular myocytes. Indeed, the authors do not exclude the possibility that the ventricular NBCn1 transcripts may originate from nonmyocytes. Nevertheless, they suggest that NBCn1 contributes to an increased intracellular Na⫹ load in hypertrophic myocytes, which would tend to reverse the Na-Ca exchanger, and thereby promote arrhythmia and reperfusion injury (1071). B) Increased protein abundance in response to hypercapnia. Chronic hypercapnia generally increases NBCn1 protein abundance in the neonatal, but not adult, mouse heart (463). C) Upregulation of NBCn1-like activity in cardiac myocytes by ANG II. In cat cardiac myocytes, 10⫺7 M ANG II stimulates HOE64268-insensitive pHi recovery from an acid-load (presumed to represent the sum of NBCe1 and

68

An NHE1 blocker.

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VI) Upper digestive system. A) Transepithelial HCO3⫺ secretion in salivary gland. The concerted action of NBCe1 and NBCn1 in the basolateral membranes of striated duct epithelia could support transepithelial HCO3⫺ secretion into the saliva ⫺ (FIGURE 21B). A suggested role for NBCn1 in apical HCO3 salvage in salivary gland duct cells lacks evidence that NBCn1 resides in the apical membrane of these cells.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Lumen

Tight junction

Interstitial fluid

Na+ NH4

ROMK

NH3

Na+

H+

NHE1

NH3

NH4+

NKCC2 2

Cl–

H+

H2O CO2

NBCn1

HCO3–

Na+

Cl–

H+

HCO3–

cAMP

AE2

mTAL epithelial cell FIGURE 34. Role of NBCn1 in the renal medulla. To avoid absorption of ammonia into the blood, NH4 traveling along the nephron bypasses the renal cortex by passing through the medullary interstitium to the ⫹ collecting tubules. NH4 enters thick ascending limb epithelia via K⫹ channels (ROMK) and the Na/K/Cl cotransporter. NH3 is absorbed across the basolateral membrane into the medullary interstitium, perhaps via ⫺ that enters the cell via NBCn1. The basolateral a channel. Residual H⫹ is extruded by NHE and titrated by HCO3 Na-pump has been omitted for clarity.

NBCn1 action, Ref. 223). However, this same dose of ANG II inhibits a DIA that is blocked by S085969 (presumed to represent isolated NBCe1 action, Ref. 224). Therefore, the authors of the study conclude that the stimulatory effect of ANG II upon NCBT activity in cat cardiac myocytes represents activation of NBCn1, rather than of NBCe1. However, although some electroneutral NCBT activity is evident in cardiac myocytes (1072), compelling evidence for NBCn1 expression in cardiac myocytes, as opposed to endothelia, is presently lacking. A pharmacological dissection of the pathway of NCBT activation in cat cardiac myocytes by De Giusti and co-workers led the authors of the study to propose that the stimulatory effect of ANG II upon NBCn1 involves activation of NADPH oxidase, generation of reactive oxygen species (ROS), ROS-induced release of mitochondrial ROS, and stimulation of the extracellular-signal regulated kinase (ERK) signaling pathway (223, 224).

69

An NCBT inhibitor of undemonstrated specificity.

III) Urinary system. A) Increased transcript and protein abundance in kidney in response to acidosis. Multiple reports demonstrate that chronic metabolic acidosis upregulates NBCn1. NBCn1 transcript abundance is increased in the rodent kidney by the oral administration of NH4Cl (207, 688),70 oral administration of HCl (207), or by the acidosis that accompanies hyperkalemia (664). Compared with wild-type controls, NBCe2-null mice exhibit a slightly greater abundance of NBCn1 transcripts and a compensated metabolic acidosis (341). At the level of NBCn1 protein, the acidosis that follows oral administration of NH4⫹ increases the levels of NBCn1 protein (530, 664), consistent with the increase of NBCn1-like activity in the isolated mTAL of these animals (694), as discussed above. NBCn1 protein levels in the mTAL also increase during the acidosis that accompanies Li⫹-induced nephrogenic diabetes insipidus (491) and hyperkalemia (431). Finally, NBCn1 protein

70 One preliminary report found that rat renal NBCn1 transcript abundance was not increased by NH4Cl feeding, but that NBCn1 protein levels were increased (664).

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Na+

H-pump H+

NHE3

++

cAMP

+

MARK D. PARKER AND WALTER F. BORON abundance in the ST-1 mTAL cell line rises following a 24-h exposure to a medium that is acidic (pH 6.8) or that contains 10 mM NH4Cl (557). B) Increased protein abundance in response to hypercapnia. Chronic hypercapnia generally increases NBCn1 protein abundance in the neonatal, but not adult, mouse kidney (463). I) CAUSES OF NBCn1 DOWNREGULATION.

Maneuvers that downregulate NBCn1 have only been reported in the brain and kidney.

B) Apparent lack of decreased protein abundance in response to alkalosis. Note that in cultured rat hippocampal neurons, raising extracellular solution to pH 8.3 does not significantly change NBCn1 protein levels, although it might be noted that the high pHo does not cause these neurons to acquire a substantially higher pHi (202). II) Urinary system. A) Decrease in protein abundance in response to ureteral obstruction. In the renal mTAL of rats in which both ureters are occluded for 24 h by tying with a silk ligature, NBCn1, together with NKCC2 (previous topic), protein abundance is substantially decreased four days after ligature release (1024). In rats in which only one ureter is obstructed within 48 h of birth, NBCn1 protein abundance is unchanged after 7 wk in the continuously obstructed kidney but is increased ⬃30% in the contralateral unobstructed kidney (1025), a pattern consistent with a compensation to the metabolic acidosis that accompanies ureteral obstruction. After 14 wk of continuous unilateral obstruction, both kidneys exhibit a ⬃20% decrease in NBCn1 protein abundance (1025), a pattern consistent with a contribution to the metabolic acidosis. B) Apparently decreased protein abundance in pendrin knockouts. Pendrin/Slc26a4 is a Cl-HCO3 exchanger that mediates the secretion of HCO3⫺ across the apical membrane of renal ␤- and non-␣/non-␤-intercalated cells (819). The physiological importance of pendrin is underlined by the observation that perfused collecting ducts from pendrin-null mice absorb rather than secrete HCO3⫺ (819). One immunohistochemical study suggests that NBCn1 protein

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C) Decreased protein abundance in response to FK506 administration. NBCn1 protein abundance falls by ⬃20% during and following the renal tubular acidosis that accompanies administration of the calcineurin inhibitor FK506 (655). Inasmuch as acidosis per se appears to increase NBCn1 protein abundance, this seemingly counterintuitive observation in FK506-treated mice probably reflects an effect of calcineurin blockade in these animals. D) Decreased protein abundance in response to alkalosis. Hypercalcemia caused by infusion of parathyroid hormone (PTH) inhibits acid secretion by the proximal tubule but causes a mild, paradoxical metabolic alkalosis and decreased urine pH. The paradox is at least in part due to an increased expression of the B1 subunit of the V-type H⫹ pump in the inner medullary collecting duct (1026). The challenge also causes a reduction in ammonium excretion. Indeed, rats treated with PTH have a ⬃60% decrease in NBCn1 protein abundance in the basolateral membranes of their mTAL and IMCD epithelia (1026). The downregulation of NBCn1 expression in the mTAL would presumably reduce urinary NH4⫹ (i.e., acid) excretion by the mechanism above. Furosemide-induced alkalosis also reduces NBCn1 protein abundance in the renal medulla of rats (754). J) CONSEQUENCES OF NBCn1 DYSFUNCTION.

Much of what we know about the pathology of NBCn1 dysfunction comes from NBCn1-null mice that, exhibiting hearing and vision loss, are a potential model of human Usher 2B syndrome. Human genetic studies have linked the SLC4A7 locus with substance abuse, neuropathy, lead accumulation, and breast cancer. These studies are considered below.

I) Central nervous system. A) Neuroprotection from glutamate cytotoxicity in a model of stroke-induced epilepsy. Ischemic injury, such as might follow a stroke, causes the release of glutamate (134) and can be associated with lowering of extracellular magnesium levels (reviewed in Ref. 635). Both glutamate addition (920) and magnesium depletion (42, 933)71 induce seizure-like activity in hippocampal neurons and are models used to study the etiology of strokeinduced epilepsy. Furthermore, glutamate can cause longterm changes in neuronal excitability and cell death (920).

71 By analogy to the inhibitory effect of intracellular Mg2⫹ on NBCe1-B, one might expect Mg2⫹ depletion to activate other AIDincluding NCBTs, such as NBCn1, which could lower the seizure threshold of hippocampal.

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I) Central nervous system. A) Decreased protein abundance in brain in response to hypoxia. As is usually the case for NDCBE and NBCn2, NBCn1 protein levels generally fall in response to chronic continuous hypoxia in the hippocampus, cerebral cortex, subcortex, and cerebellum of neonatal and adult mice (174). One possibility for these effects is that the hypoxia downregulates a range of energy-requiring systems, including NCBTs. Another is that the hypoxia triggers hyperventilation and thus respiratory alkalosis, which indirectly causes a downregulation of NCBTs.

levels are decreased in the cortical collecting ducts of pendrin-knockout mice, particularly in those cells that usually express pendrin (492). However, as discussed above and in Appendix VII, the anti-NBC3 antibody used in this study yields results that conflict with those obtained using other methods of detecting NBCn1 protein.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

In cultures of mouse hippocampal neurons, NBCn1 knockdown is neuroprotective inasmuch as fewer NBCn1-null than wild-type neurons die when glutamate is applied in the nominal absence of extracellular Mg2⫹ (202). The neuroprotective role of NBCn1 knockdown in these experiments is consistent with the hypothesis that a reduction in pHi reduces neuronal excitability and is also in accordance with the higher seizure threshold of brain slices from NDCBE and NBCn2 knockout mice (429, 889), the enhanced neuronal survival in NHE1-null mice following ischemic injury (614), and the proposed enhancement of neuronal excitability by NBCe1.

C) Genetic linkage with propensity towards substance abuse. A chromosomal locus associated with a high degree of allelic variation in substance abusers includes the SLC4A7 gene. Ishiguro and co-workers (423) studied the association of addictive behavior with the frequency of occurrence of 22 singlenucleotide polymorphisms (SNPs) in the SLC4A7 gene region. Of these 22 SNPs, 12 occurred with a significantly increased frequency in genomic DNA samples from substance abusers compared with control samples. Of these 12 SNPs, 5 are located in exons but only one, designated rs3755652, changes the predicted coding sequence of the NBCn1 protein, producing a Glu to Lys mutation midway through splice cassette II. The effect of any of these SNPs on the functional expression of NBCn1 activity is untested. Ishiguro et al. note that far more than 22 SNPs may need to be examined in the gene region,72 and they do not exclude the importance of SNPs in neighboring genes. Furthermore, SLC4A7 was not sequenced in its entirety for mutations. Thus the role, if any, of NBCn1 in the etiology of addictive 72 Indeed, as of May 2012, the number of allelic SLC4A7 variations in the NCBI SNP database stands at 1449, 85 of which are located in exons and 46 of which alter the predicted NBCn1 coding sequence.

II) Sensory organs. A) Vision and hearing loss: a potential model of Usher syndrome. One strain of NBCn1 knockout mice develop blindness and auditory impairment due to the degeneration of photoreceptors in the retina (93) and of hair cells in the inner and outer ear (93, 607). The signs manifested in the knockout mouse are similar to those of Usher syndrome 2B, a disease once linked to the human chromosomal locus 3p23–3p24.2 (383), which is close to the location of the human SLC4A7 gene. However, the original assignment of an Usher locus at 3p23–24 has since been retracted (384). Ironically, molecular evidence gathered in the interim suggests that NBCn1 could be part of the Usher protein network (790), disruption of which is considered to be the molecular basis of Usher syndrome (789). In the absence of a clear demonstration that mutations in human SLC4A7 gene are linked to Usher syndrome 2B, the Slc4a7-null mouse remains only a potential model of the human disease. In a screen of 172 individuals with Usher syndrome, no mutations localized within the exons that encode the NBCn1-A product (549). This observation does not exclude the possibility that disease-associated mutations could be located in the promoter, introns, or exons included in NBCn1 variants besides NBCn1-A (e.g., those that include cassette III). However, the majority of the 172 individuals in the study exhibited mutations in known Usherassociated genes (549). B) Genetic linkage with central cornea thickness in mice. Strains of mice with thicker corneas tend to exhibit, in their corneas, greater abundance of certain transcripts, including those encoded by Slc4a7 (601). Corneal thickness in the studied mouse strains is mostly determined by the number of lamellae in the corneal stroma, an observation presumed to be due to altered keratocyte function (601). The role of NBCn1 in this likely complex phenotype, has not been determined. III) Peripheral nervous system. A) Possible role in hereditary sensory neuropathy. The human SLC4A7 gene locus falls within the boundaries of a region (3p24) that has been linked to a mild sensory neuropathy associated with a chronic cough and gastroesophageal reflux (505). In their 2004 study, Kok et al. (504) sequenced the coding exons of SLC4A7 from genomic DNA amplified from the white blood cells of at least one affected individual and found no nonsynonymous mutations. On these grounds alone, the authors exclude SLC4A7 as a candidate gene for the neuropathy. However, this study does not identify nonsynonymous mutations in the coding exons of any other genes in this candidate region and does not consider the possibility that causal mutations may be located in regulatory regions

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B) Possible contribution to enhanced NMDA-associated neurotoxicity in acidosis. NMDA, an agonist for the NMDA class of ionotropic glutamate receptors, causes cell death through excitotoxicity. As judged by caspase-3 activation, NMDA-induced neuronal death is greater in acidotic rats than in wild-type rats (709). Although upregulation of NBCn1 in acidotic rats normally counters intracellular acidosis, NBCn1 is not upregulated by acidosis in NMDA-treated rats (709). The authors suggest that the lack of enhanced NBCn1 activity might render neurons more susceptible to acid injury in NMDA-treated rats, resulting in increased cell death (709). This hypothesis is consistent with the anti-apoptotic effect of NBCn1, apparently mediated by a rise in pHi, in CSF1-stimulated osteoclasts. An alternative explanation provided by those authors is that NMDA is killing the cells before they have a change to upregulate NBCn1 (709). It is unknown how other neuronally expressed NCBTs are affected by NMDA treatment in this model.

behaviors is unknown. However, the association seems reasonable, given the potential contribution of NBCn1 toward control of neuronal excitability.

MARK D. PARKER AND WALTER F. BORON of the SLC4A7 gene or in additional genes outside of 3p24. Therefore, it is premature to exclude a role for SLC4A7 in the physiopathology of this syndrome.

NBCn1-null mice are mildly hypertensive at rest, an observation that accords with the finding that isolated, precontracted arteries from these mice exhibit a reduced ability to relax in response to acetylcholine application (92). Underlying this phenotype is reduced pHi in vascular cells. Moreover, endothelial nitric oxide synthase (eNOS) is inhibited by acidosis in endothelial cells, whereas rho-kinase signaling is inhibited in vascular smooth muscle cells, rendering contraction of isolated arteries less sensitive to Ca2⫹ (92). B) Suggested linkage with blood lead accumulation. A genetic linkage study suggests a quantitative trait locus for erythrocyte lead accumulation, with a linkage peak near the gene locus of 62 genes or putative genes, including human SLC4A7 (1033, 1034). NBCn1 is the only transporter encoded by any of these 62 genes, leading the authors to conclude that NBCn1 affects lead transport. However, this conclusion must be regarded with caution because the authors present no evidence that 1) erythrocytes express NBCn1,73 2) NBCn1 mediates lead transport, or 3) mutations in SLC4A7 affect lead transport in any cell type. On a related note, the author of one study on red blood cells reports evidence consistent with AE1-mediated transport of PbCO3⫺ (886). V) Lower digestive system. A) Potential role in susceptibility to duodenal ulcers. Helicobacter pylori markedly inhibits the ability of the duodenal epithelium to increase HCO3⫺ secretion in response to the appropriate stimuli (984), leading to duodenal ulceration (reviewed in Ref. 639). Because NBCn1 action supports duodenal HCO3⫺ secretion, NBCn1 defects could increase susceptibility to duodenal ulceration. VI) Reproductive system. A) Linkage to breast cancer. An association between NBCn1 and cancer was first broached

In the first study that linked cancer with SLC4A7, Chen and co-workers (182) identified NBCn1 as a tyrosine kinase substrate expressed in the lobular acini of the breast. The authors make two observations that link NBCn1 to cancer. 1) In the MCF10AT cell line model of breast cancer progression, NBCn1 tyrosine phosphorylation was increased threefold in premalignant and low-grade-lesion-like cells but was decreased twofold in high-grade-lesion-like cells (182). However, the effect of phosphorylation events on the functional expression of NBCn1 activity in tumor cells is untested. 2) NBCn1 protein abundance is decreased in MCF10AT cells and in most of the cancerous breast-tissue samples examined in their study. However, the downregulation of NBCn1 as a contributory factor in breast cancer seems counterintuitive: NBCn1 expression would help cancer cells to maintain a normal pHi in the acidic environment of a tumor and would enhance local extracellular acidity. Other studies provide evidence that cancer is associated with upregulation of NBCn1. The breast-cancer cell line MCF-7, when overexpressing a truncated ErbB2 receptor, exhibits enhanced acid-extruding capability in part by increasing the abundance of NBCn1 protein in the plasma membrane (546). A report by Wong et al. (1041) demonstrates the importance of NCBT activity, including a DIDSinsensitive component, to pHi regulation in two human and one murine breast-cancer cell lines. Note that both groups discount a significant role for NBCn1 action in cancer cell migration (547, 849), although in theory NCBT activity could contribute to a regulated volume increase. In summary, the role of NBCn1 in the etiology of cancer progression is presently speculative, although NBCn1 abundance and phosphorylation may be useful markers for cancer screening (182). 2. NDCBE (Slc4a8)

73 SLC4A7 products are not among the 340 red cell proteins identified in a proteomic study by Pasini and co-workers (727), nor among the 751 proteins reported in a review of the human red cell proteome (329).

894

The electroneutral Na⫹-driven Cl-HCO3 exchanger NDCBE (encoded by the Slc4a8 gene) exchanges Na⫹ and two HCO3⫺ equivalents for Cl⫺, a function shared by A) SUMMARY.

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IV) Circulatory system. A) Genetic linkage with hypertension. A genome-wide association study (GWAS) links a single nucleotide polymorphism, rs13082711, in the SLC4A7 gene locus with slightly elevated systolic and diastolic blood pressure in individuals of European and African ancestry (961). Inasmuch as NBCn1 is a Na⫹ transporter that is expressed in the vasculature and kidney, the protein has the potential to influence blood pressure. However, the SNP is located ⬃10 kb upstream of any known transcriptional start site for NBCn1, and the effect of this SNP, or of yet to be discovered SNPs in the linked region, upon NBCn1 expression has yet to be established.

in a 2003 review by Izumi et al. (426). Since then, 10 studies have been published concerning the link between susceptibility to breast cancer and a genetic locus marked by an SNP, rs4973768, that is located in the long terminal exon that encodes the 3=-UTR of NBCn1 (12, 44, 148, 182, 364, 605, 648, 670, 733, 917). It is important to note that it is the genetic locus marked by this SNP, rather than the SNP itself, that is linked to breast cancer susceptibility. Thus it is possible, as noted by the authors of these studies, that dysregulation of the neighboring NEK10 gene, a UV-stimulated kinase that is independently associated with cancers (656), could underlie the genetic susceptibility.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

NBCn2 under certain conditions. NDCBE has four known variants (-A through -D). In common with several other NCBTs, NDCBE-B is stimulated by the soluble protein IRBIT. Although present in many organs, NDCBE is notably abundant in the brain. In neurons, NDCBE-mediated HCO3⫺ influx enhances neuronal excitability, a role corroborated by a study of NDCBE-null mice. To date, no human pathologies have been linked to NDCBE dysfunction.

C) MOLECULAR ACTION OF NDCBE.

When expressed in Xenopus oocytes, human NDCBE mediates electroneutral codependent Na⫹ and HCO3⫺ influx (i.e., Na⫹ influx requires HCO3⫺ and vice versa), accompanied by a Na⫹ and HCO3⫺dependent Cl⫺ efflux (337). When NDCBE operates in the “reverse” direction, that is to say, mediating the coefflux of Na⫹ and HCO3⫺ from the oocyte, the transport process has an absolute dependence on extracellular Cl⫺ (337). Thus NDCBE is a Na⫹-driven Cl-HCO3 exchanger (FIGURE 2⫺ 35A). A preliminary study suggests that it is CO3 and not ⫺ HCO3 that is the transported base (Fig. 35, B and C; Ref. 335). An NCBT activity attributed to NDCBE in mouse IMCD cells is poorly selective for Na⫹ over Li⫹ (35). The approximate stoichiometry of transport is estimated to be 1Na⫹:2HCO3⫺, which would require the net countertransport of 1 Cl⫺ for electroneutrality (337). Inasmuch as 1) the estimated unidirectional efflux of 36Cl is manyfold greater than the estimated fluxes of Na⫹ and HCO3⫺ (337) and 2) the NDCBE-mediated efflux of Cl⫺ has a trans-side Cl⫺ dependence (719), it seems likely that the net movement of chloride by NDCBE is accompanied by a much larger component of futile Cl-Cl self-exchange (FIGURE 35D; Ref.

74 This partial human clone (AF107099) is identical along its length to subsequently cloned human SLC4A8 products. A full-length mouse Slc4a8 product (now called NDCBE-A) was subsequently cloned in its entirety by a group that included the same authors (1029). 75 GenBank nucleotide accession number AF069512.

I) Assignment of transport activity to NDCBE. It is straightforward to overexpress NDCBE in a cell such as a Xenopus oocyte and convincingly demonstrate NDCBE activity. However, because of the vagaries of Cl⫺ transport, it can be a challenge to demonstrate that transport activity is indeed due to NDCBE in a setting where the transporter coexists with other NCBTs, Cl-HCO3 exchangers (including those from the Slc26 family), Na-H exchangers, and Cl⫺ channels. Thus reports of NDCBE activity, hereafter referred to as NDCBE-like activity, can rarely be taken at face value. To illustrate, we will note the potential difficulties in using three common approaches to test for the presence of NDCBE activity. A) 36Cl fluxes. An NDCBE should mediate an efflux of 36Cl that requires extracellular Na⫹ and HCO3⫺, and that is blocked by DIDS. As discussed in a later section, the human electroneutral Na/HCO3 cotransporter NBCn2, as expressed in oocytes, does not normally mediate net Cl⫺ transport, but is nonetheless capable of futile Cl-Cl exchange (detected as 36Cl efflux) that requires HCO3⫺ but not Na⫹, and that is blocked by DIDS. It is not known whether the closely related NBCn1 can also mediate futile Cl-Cl exchange. Thus a convincing demonstration of NDCBE activity requires evidence of net Cl⫺ efflux, either from a direct and quantitative comparison 36Cl influx and 36Cl efflux, or from surface-[Cl⫺] transients as discussed below. Further complicating matters, in the absence of extracellular Cl⫺, NBCn2 appears capable of Na⫹-driven Cl-HCO3 exchange. Even the red cell anion exchanger AE1 has been reported to be capable of exchanging Cl⫺ for either the NaCO3 or the LiCO3⫺ ion pairs under certain conditions (303, 304). B) Net Cl⫺ fluxes. An NDCBE should mediate a net efflux of Cl⫺. However, in cells that express a Cl⫺ channel plus NBCe1 or NBCe2, the coupled influx of Na⫹, HCO3⫺, and net negative charge would hyperpolarize the cell and thus drive the net efflux of Cl⫺ through the Cl⫺ channel. Voltage-clamp experiments could test this possibility. C) Washout of intracellular Cl⫺. The net influx of Na⫹ and HCO3⫺ mediated by an NDCBE should require intracellular Cl⫺. Unfortunately, it is notoriously difficult to wash Cl⫺ out of cells (115, 850, 851). Moreover, human NDCBE appears to require extracellular Cl⫺ for NDCBE activity (719). Finally, as noted above, NBCn2 appears to act as a Na⫹driven Cl-HCO3 exchanger in the absence of extracellular Cl⫺. Thus removing extracellular Cl⫺ for the purpose of Cl⫺ washout could have unintended consequences for key NCBTs. Thus a demonstration of a pHi recovery from an acid load, together with dependence on Na⫹ and HCO3⫺ but blockade by DIDS, is only the beginning of a physiological assignment of NDCBE. The next critical step is to demonstrate net

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B) NOMENCLATURE OF SLC4A8 PRODUCTS. Following the provisional assignment of NBC1 (now called NBCe1) to refer to Slc4a4 products, and the provisional assignment of NBC2 (now called NBCn1) to refer to Slc4a7 products, two groups simultaneously reported the cloning of two different Slc4 products to which they inadvertently assigned the degenerate name NBC3. We now appreciate that the “NBC3” reported by Pushkin et al. (765) is an Slc4a7 product (139), whereas the “NBC-3” reported Amlal et al. (35) is a partial human Slc4a8 product.74 In fact, the report of the partial sequence postdated the depositing of a full-length human SLC4A8 product by Grichtchenko et al.75 With the physiological characterization of the SLC4A8 product as a Na⫹driven Cl-HCO3 exchanger, the product was renamed NDCBE1 (337). With the assumption that the SLC4A8 gene encodes the sole human Na⫹-driven Cl-HCO3 exchanger, we propose to drop the numerical suffix, and refer to the transporter as NDCBE.

337). Moreover, this Cl-Cl self-exchange has an absolute requirement for extracellular Na⫹ and HCO3⫺ (337, 719).

MARK D. PARKER AND WALTER F. BORON Na+ CO32–

NDCBE Na+

HCO3– HCO3–

A

Na+

CO32–

B

Cl–

(n) Cl–

NaCO3–

C

Cl–

D

Cl–

(n+1) Cl–

FIGURE 35. Molecular action of NDCBE. Possible molecular mechanisms by which NDCBE could operate in ⫺ an electroneutral mode to exchange Na⫹ and HCO3 equivalents for Cl⫺. Note that we have not considered any 2⫺ /H⫹ cotransport. In the original characterization of NDCBE action, Cl⫺ flux was models that are based on CO3 estimated to be sixfold greater than Na⫹ flux.

D) THE SLC4A8 GENE. The human SLC4A8 gene maps to 12q13 (337, 673), locus 12q13.13 in version 36.3 of the NCBI human genome map, and includes at least 28 exons spread over 124 kb (FIGURE 36A; Ref. 717). SLC4A8 is located between GALTNT6 (that encodes UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) and SCN8A. We discuss alternative splicing of SLC4A8 premRNA in the following section. The intrinsic promoter activity of a virally derived long-terminal repeat sequence LTR129, present normally in intron 5 of the human SLC4A8 gene, is activated in testicular cancer. The transcript promoted by the LTR is a short antisense-SLC4A8 intronic sequence. However, this antisense pre-mRNA does not appear to affect NDCBE transcript abundance in seminoma versus normal testicular parenchyma (143). However, when overexpressed in a testicular carcinoma cell line, the antisense sequence does have the capability to reduce NDCBE transcript abundance, acting at the level of pre-mRNA (327). E) STRUCTURAL FEATURES AND VARIANTS OF NDCBE. The human SLC4A8 gene has the capacity to encode at least five variant products named A–E (FIGURES 36, B and C, and 37). Evidence for other minor variants has also been reported, including NDCBE-D=, which has an alternative 5=-UTR. NDCBE-A and NDCBE-B share a common Nt, but NDCBE-A has a longer and different Ct. NDCBE-C and NDCBE-D are identical to “A” and “B,” respectively, but their Nt are truncated by 54 amino acid. NDCBE-E has a longer and different Nt appendage compared with NDCBE-B. Protein variants A–D exhibit NCBT function

896

when expressed in Xenopus oocytes; NDCBE-E ought to be functional but has not been tested. The choice of Nt has no obvious bearing on basal functional expression of NDCBE. However, variants with the shorter Ct (i.e., NDCBE-B and -D) show reduced functional expression compared with variants with the longer Ct (i.e., NDCBE-A and -C). A comparison of the functional expression of NDCBE-A and NDCBE-B with an artificial construct that includes neither the 17-amino acid nor the 66-amino acid sequence (FIGURE 37) demonstrates that the 17-amino acid sequence is inhibitory to the functional expression of NDCBE. The 66-amino acid sequence has no effect on the basal functional expression of NDCBE (717). I) Sources of variation in coding sequence among NDCBE variants. Known NDCBE variants differ only in the length of their Nt and the choice of one of two Ct appendages. Unique among NCBTs, transcripts that encode each Ct include mutually exclusive 3=-UTR regions. A) Alternative promoter choice and truncated Nt. Some variants of NDCBE are truncated by 54 amino acid in their Nt as a result of alternative promoter choice (717). The SLC4A8 gene appears to have two promoters (FIGURE 36C). One promoter (P1) is located just upstream of exon 1 and promotes transcription from exon 1. Transcription initiated at exon 1 produces pre-mRNAs that can be processed to form either NDCBE-C, NDCBE-D, or NDCBE-E. Because exons 2– 4 are omitted from NDCBE-C/D transcripts and because neither exon 1 nor exon 5 contains an initiator Met, translation of NDCBE-C/D is predicted to begin with an initiator Met located within exon 6 (that encodes internal Met55 of NDCBE-A/B). Thus in NDCBE-C/D the first 54 amino acids of NDCBE-A/B are absent. The consequence of the loss of this Nt sequence are currently unclear, but preliminary data suggest that unlike NDCBE-B (722), NDCBE-D may not be sensitive to stimulation by IRBIT due to the loss of sequence homologous to that which contains IRBIT binding determinants in NBCe1-B (Parker and Boron, unpublished data). Translation of NDCBE-E is predicted to begin with an initiator Met located within exon 3.

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Cl⫺ efflux under conditions in which Vm does not change or in a cell verified to be devoid of electrogenic NBCs or Cl⫺ channels. Finally, it is advisable to demonstrate the presence of NDCBE protein at the plasma membrane, and show that knock-down of the protein eliminates the hypothesized NDCBE activity. With these caveats in mind, we summarize the reports of Na⫹-dependent Cl-HCO3 exchange activity and indicate, where possible, which reports are strongly linked to the accompanying presence of NDCBE itself and which are unlikely to involve NDCBE.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

A

Locus 12q13 20 kb GALNT6

B

SLC4A8

SCN8A

Gene structure 10kb P2

P1

1

C

2-3

4

5

25

6

28

Transcript variation P2 M

4

NDCBE-A

5

6

7

24

5

6

7

24

6

7

24

M

4

NDCBE-B

25 a

26

27

* 28

26

27

* 28

* 25

M

25 a

NDCBE-C

1

5

NDCBE-D

1

5

6

7

24

* 25

NDCBE-D

1

5

6

7

24

* 25

M

M

2

3

FIGURE 36. SLC4A8 gene structure and NDCBE transcript variants. Scale diagrams showing the human SLC4A8 gene locus together with the position of neighboring genes (A), the position of promoters (P1 and P2), and the position of exons within SLC4A8 (B). Transcript variants are represented, not to scale, as numbered boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature transcript. “//” denotes that all five transcripts include exons 7–24. Exons that include the initiator ATG codon (“M”) and termination codon (“*”) are marked for each transcript. Sequences that are derived from part of a larger exon sequence are labeled with an “a” (e.g., exon 25a is a subdivision of exon 25). Colored exons, or parts of exons, correspond to the protein regions that each encodes, which are identically colored in FIGURE 37. Uncolored exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line are predicted, but not demonstrated, to be included in the mRNA.

A second promoter (P2) is situated upstream of exon 4 and promotes transcription of NDCBE from exon 4, which encodes an initiator Met. Transcription from this promoter produces pre-mRNAs that can be processed to form either NDCBE-A or NDCBE-B. B) Alternative Ct and 3=-UTR. NDCBE variants can have either a 17-amino acid or a 66-amino acid sequence appended to the ⬃30 amino acid that is common to the Ct of all NDCBE variants. A 66-amino acid Ct appendage is produced when exons 25a-28 are spliced together, making exon 28 the terminal exon (FIGURE 36C). The 66amino acid appendage (encoded by exons 26 –28) is common to NDCBE-A and NDCBE-C. The role of the 66amino acid Ct is presently unknown; this 66-amino acid Ct can be removed without any apparent consequence to functional expression of the transporter in Xenopus oocytes (717).

An alternative mRNA is produced when the splice machinery does not recognize the exon-25a/intron-25 splice boundary and a polyadenylation signal located ⬃3 kb downstream of the start of exon 25 is used to produce a mature, polyadenylated mRNA. This “long” version of exon 25 is defined as a composite terminal exon (268). The 17-amino acid appendage (encoded by exon 25) is common to NDCBE-B, -D, and -E and constitutes an autoinhibitory domain, inasmuch as a mutant NDCBE that lacks the 17amino acid sequence has a greater functional expression than an NDCBE that includes the 17-amino acid sequence (717). The 3=-UTR of NDCBE-B/D/E (comprised of exon 25 sequence) is different from and shorter than that of NDCBEA/C (comprised of exon 28 sequence), probably accounting for the two groups of transcript sizes (9.5–12 kb and 4.4 – 6.3 kb) observed in Northern blots (35, 337, 717). This

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P1

MARK D. PARKER AND WALTER F. BORON Nt

TMD 1–5

100 aa

6–9

Ct 10–14

NDCBE-A

66 16

1,093

AID 1,044

NDCBE-B 17

16 NDCBE-C

∆54

NDCBE-D

∆54

66

1,040

991 17 1,071

43

17

FIGURE 37. NDCBE protein variants. Scale diagram of protein variants that are encoded by the transcripts represented in FIGURE 36C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino acids and colored to denote their genetic origin as shown in FIGURE 36C. A color-matched protein sequence alignment of the variants is provided in Appendix V.

choice of alternative 3=-UTRs is a mechanism of variation that is unique among SLC4 products.

NDCBE-C has been cloned from human brain, heart, and kidney cDNA preparations (717).

C) Cloned NDCBE variants that are demonstrated or likely to exhibit NCBT activity. There are five NDCBE protein variants, the features of which are described below and depicted in FIGURE 37. GenBank protein accession numbers for the variants discussed in this section are provided in Appendix IV.

4) NDCBE-D and -D= (NCBT activity demonstrated). NDCBE-D is identical to NDCBE-B, except that it does not include the first 54 amino acids of the NDCBE-B Nt. NDCBE-D includes the autoinhibitory 17-amino acid Ct appendage. NDCBE-D protein is the shortest of the four variants. Full-length NDCBE-A has been cloned from human brain and kidney cDNA preparations (717). NDCBE-D= is identical to NDCBE-D, except for a 5= extension to exon 6, which extends the 5=-UTR and is specifically expressed in the heart. The relevance of the 5=-UTR extension is presently unclear (717).

1) NDCBE-A (NCBT activity demonstrated). Human NDCBE-A is the counterpart of the archetypal mouse NDCBE variant that was reported in Reference 1029. NDCBE-A includes the full-length Nt and the 66-amino acid Ct appendage. NDCBE-A protein is the longest of the four variants. Full-length NDCBE-A has been cloned from a mouse renal cell line (1029) and human brain (717) cDNA preparations. 2) NDCBE-B (NCBT activity demonstrated). NDCBE-B is the archetypal human NDCBE clone reported in Reference 337. NDCBE-B includes the full-length Nt and the autoinhibitory 17-amino acid Ct appendage. As a consequence, NDCBE-B has a lower per-molecule activity than NDCBE-A when expressed in Xenopus oocytes. Fulllength NDCBE-B has been cloned from human brain cDNA preparations (337, 717). 3) NDCBE-C (NCBT activity demonstrated). NDCBE-C is identical to NDCBE-A, except that it does not include the first 54 amino acids of the NDCBE-A Nt. NDCBE-C includes the 66-amino acid Ct appendage. Full-length

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5) NDCBE-E (NCBT activity untested). A singleton cDNA from brain (GenBank DNA accession no. AB018282) that appears to represent a full-length mRNA would, if translated, produce a protein product in which the 16 amino acids encoded by exon 4 of NDCBE-A/B are replaced by 43 amino acids encoded by exon 3. Such modification of the Nt appendage is unlikely to eliminate NCBT activity and thus NDCBE-E is likely to be functional. D) Predicted NDCBE variants. 1) Partial clones from human cDNA. A number of other human NDCBE cDNA sequences that have been deposited in GenBank have a structure similar to NDCBE-C/D, in that as their transcription begins at a position that is upstream of, but omits, exon 2. If a full-length NDCBE-C/D clone was modified to include any of these partial sequences (e.g., GenBank DNA accession nos. CN286464

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NDCBE-E

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

and DB090766), the altered sequence of the Nt appendage ought not eliminate NCBT function. E) Other NDCBE variants. 1) An unusual variant that represents only the isolated Nt. A singleton NDCBE cDNA amplified from thymus (GenBank DNA accession no. AK128321) includes exons 4 –11, and exon 12, which becomes a composite terminal exon that includes intron 12 sequence and a polyadenylation signal therein. Such a transcript would encode residues 1–314 of NDCBE-C/D plus 15 novel amino acids encoded by the exon 12 extension, followed by a termination codon: that is, most of the soluble Nt of NDCBE-C/D. It is not clear whether this protein product, truncated within the Nt, would be stable. This cDNA is reminiscent of isolated Nt variants of NBCn1 and NBCn2.

2) Putative variants cloned from rodent cDNA (potentially legitimate transcripts, NCBT activity unlikely). Mouse Slc4a8 encodes NDCBE-A, but it is unknown whether the gene has the capability to encode orthologs of NDCBE-B, -C, or -D. The rat Slc4a8 gene has not been demonstrated to produce orthologs of any of the four human variants. The three reported transcripts from rat kidney are named “NDCBE1-A,” “NDCBE1-B,” and “NDCBE1-C” and appear to be unique to rat and are not the same as the human NDCBE-A/B/C variants. In fact, none of the three rat clones is predicted to encode a functional transporter. Rat “NDCBE1-A” lacks putative TM6 and part of putative TM7. Rat “NDCBE1-B” lacks 25 amino acids in the cytoplasmic Nt close to TM1; at least for NBCe1, this sequence is necessary for functional expression of NCBT activity (575). Rat “NDCBE1-C” lacks both of the regions missing from “NDCBE1-A” and “-B”. The rat Slc4a8 gene does have the potential to encode a complete ortholog of the human NDCBE-A variant, but cDNA representing such a transcript has yet to be cloned. F) DISTRIBUTION OF NDCBE. NDCBE expression is particularly abundant in brain, specifically in neurons, although NDCBE transcripts are also abundant in the testis and are expressed to a lesser extent in many other organs. The distribution of NDCBE in specific organ systems is discussed below. The distribution of NDCBE is summarized and compared with that of other NCBTs in TABLE 4.

I) Central nervous system. A) Brain. In Northern blots and RT-PCR analysis of mouse and human RNA preparations,

At the regional level, NDCBE-A/B transcripts are present in RNA preparations from amygdala, caudate nucleus, cerebellum, cerebral cortex, corpus callosum, hippocampus, medulla, substantia nigra, and thalamus (337, 1029). RTPCR amplifies NDCBE, as well as NBCe1 and NBCn1, transcripts from basal ganglion, occipital cortex, hypothalamus, and frontal lobe (327). The widespread distribution of NDCBE throughout the CNS of rats and mice is confirmed by the use of anti-NDCBE antibodies (553, 889). The use of a pan-NDCBE antibody further extends the distribution of NDCBE to the entorhinnal cortex, midbrain, striatum, pons, thalamus, and olfactory bulb of rats (553). An anti-NDCBE-A/C antibody does not detect NDCBE in the corpus callosum of mice (889). At the cellular level, and in brain slices, anti-NDCBE-A/B antibodies detect NDCBE in hippocampal pyramidal neurons of human (214), rat, and mouse (176; decreasing in abundance from CA1 to CA3) and in cerebellar Purkinje cells of rat (214) and mouse (176). In mouse brain slices, NDCBE-A/B is further detected in cerebellar granule cells, white matter, substantia nigra, and neurons of the brain stem (176) as well as in unipolar brush cells and cartwheel cells (interneurons) in the dorsal cochlear nucleus and in unipolar brush cells in the cerebellum (489). NDCBE-A/B is mainly expressed in neurons, as evidenced by staining of brain slices, freshly dissociated neurons, and cultured neurons, and does not have a substantial astrocytic presence (176, 889). However, a Cl⫺-dependent NCBT activity detected in cultured rat cerebellar astrocytes (500) may be attributable to an alternative Slc4a8, or even an Slc4a10, gene-product. At the subcellular level, NDCBE is abundant throughout the cell body, and to a lesser extent the processes, of hippocampal pyramidal neurons of rats (553). NDCBE-A/C immunoreactivity is predominantly localized to the presynaptic nerve endings of glutamatergic neurons where it is colocalized with glutamate transporters (136, 889), with only a marginal presence in GABAergic neurons (136, 889). B) Spinal cord. Northern blots detect the presence of NDCBE transcripts in human spinal cord preparations (35), a result not duplicated using an NDCBE-A/B specific probe (337) perhaps indicating that NDCBE-C/D are prevalent here. Indeed, an antibody that should recognize all NDCBE variants detects NDCBE protein in protein preparations from rat spinal cord (553).

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A polyadenylation signal has been identified in intron 5 of SLC4A8 (see Supplemental Table 1 of Ref. 970). The existence of a transcript composed of exon 4 and a composite terminal exon 5 has not been demonstrated, but such an mRNA could, for example, encode amino acid residues 1– 44 of NDCBE-B plus the 7-amino acid sequence “GKNCHAV” followed by a termination codon. The function, if any, of such a polypeptide is unknown.

of those organs tested, NDCBE transcripts are particularly abundant in the brain (35, 214, 337, 673, 684, 1029). In Northern blots of human brain shown in Reference 337, probed with oligonucleotide specific for NDCBE-A/B, a ⬃12 kb transcript (likely NDCBE-A) predominates over a ⬃6.3 kb transcript (likely NDCBE-B).

MARK D. PARKER AND WALTER F. BORON C) Choroid plexus. RT-PCR amplifies NDCBE, as well as NBCe1 and NBCn1, cDNAs from human choroid plexus preparations (214) but not from adult mouse or rat CPE (755). NDCBE-A/B protein is however, expressed at the basolateral membrane of choroid plexus epithelia in fetal, but not adult, rats (176). II) Sensory organs. We are not aware of any reports of NDCBE expression in the eye, ear, or olfactory system. However, NDCBE-like activity has been reported in mammalian lens cells (33, 265).

IV) Respiratory system. A) Trachea. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the human trachea (35). B) Lung. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the lungs of mice (1029) and humans (35). NDCBE transcripts are also detected in a Calu-3 human airway epithelia cell line (515). V) Circulatory system. A) Heart. NDCBE-C and NDCBE-D= transcripts can be amplified from human heart cDNA, and the significant presence of NDCBE transcripts in mouse ventricle preparations has been confirmed by qPCR (31). B) Vasculature. NDCBE cDNAs have been amplified from preparations of mouse aorta (571). VI) Musculoskeletal system. A) Skeletal muscle. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in human skeletal muscle (35). VII) Upper digestive system. A) Stomach. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the human stomach (35).

X) Endocrine system. A) Widespread. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the thyroid glands of mice (571) and in the thyroid and adrenal glands of humans (35). XI) Urinary system. A) Kidney. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the kidneys of mice (1029) and humans (35). In rat kidney, Northern blotting reveals that NDCBE transcripts are enriched in the medulla compared with the cortex (1029), a result confirmed by RT-PCR from human RNA preparations (214). In rat kidney, NDCBE transcripts predominate in the inner medulla (986, 1029), with lower abundance in outer medulla (1029). RT-PCR also detects NDCBE transcripts in a mouse IMCD-3 cell line from the inner medullary collecting duct (1029). Immunocytochemistry using an antibody directed against an epitope common to NDCBE-A and -B has not demonstrated the presence of NDCBE protein in any renal structure except endothelial cells (214), whereas an antibody directed against an epitope common to NDCBE-A and -C exhibits strong immunoreactivity in mouse renal cortical preparations, including isolated CCD preparations (571). Thus it is possible that renal epithelia predominantly express NDCBE-C and/or -D, or novel NDCBE variants.

VIII) Lower digestive system. A) Widespread. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the human pancreas and liver (35). RT-PCR analysis confirms the presence of NDCBE transcripts in the pancreas and extends this distribution to include the human duodenum, ileum, and colon (214).

XII) Reproductive system. A) Male. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the human testis (337). RT-PCR analysis confirms the presence of NDCBE transcripts in rat testis (214) and extends the distribution, in mice, to testis, epididymis, and vas deferens (599).

IX) Lymphatic and immune systems. A) Widespread. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the bone marrow and lymph nodes of humans (35). PCR analysis extends this distribution to include a T-cell-derived cell line (983).

B) Female. NDCBE transcripts have been detected by RTPCR of mouse oocytes, ovary, uterus, and vagina (275, 599). According to an NCBI-curated database of ESTs, NDCBE transcripts may be abundant in human mammary gland preparations (Appendix VI).

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III) Peripheral nervous system. A) Possible presence in trigeminal ganglion neurons. Although a preliminary study suggested the presence of NDCBE transcripts in trigeminal ganglion neurons (407), a later single-cell PCR study by the same authors was negative for NDCBE in these cells (408).

An NDCBE-like activity has been described in rat lymphocytes. Stakisaitis et al. (903) report that Na⫹-dependent ClHCO3 exchange is responsible for the net Cl⫺ efflux, measured as a fall in [Cl⫺]i, observed when cells are bathed in a solution lacking Cl⫺ (903). However, this result is not consistent with the phenotype of human NDCBE heterologously expressed in Xenopus oocytes: human NDCBE mediates a 36 Cl efflux only in the presence of extracellular Cl⫺ (719). The apparent discrepancy between the lymphocyte and oocyte data could represent systematic differences between rat versus human NDCBE, or between native lymphocytes versus heterologous expression in oocytes. However, it is possible that the Cl⫺ efflux observed by Stakisaitis et al. in rat lymphocytes is mediated by NBCn2. Expression of NDCBE and/or NBCn2 in lymphocytes has, to our knowledge, never been formally demonstrated.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

C) Placenta. Northern blots of RNA preparations reveal the presence of NDCBE transcripts in the human placenta (35). G) PHYSIOLOGICAL ROLES OF NDCBE.

NDCBE likely contributes to pHi regulation in all of the cell types in which it is expressed. In neurons, pHi, and therefore NDCBE action, influences neuronal excitability. In epithelia, NDCBE has been suggested to contribute to HCO3⫺ secretion and Cl⫺ reabsorption.

II) Central nervous system. A) Enhancement of neuronal excitability. In 1992, Church (194) found that the switch from a CO2/HCO3⫺-free HEPES buffer to a CO2/HCO3⫺ buffer is associated with enhanced neuronal excitability in CA1 neurons of rat hippocampal slices. He suggested that excitability increases because CO2/HCO3⫺ causes pHi to rise. Later Bevensee et al. (78) demonstrated that CA1 neurons in fact exist in two resting pHi states, those with a relatively low and those with a relatively high pHi (78). In those with a relatively low initial pHi in a CO2/HCO3⫺-free buffer, the switch to CO2/HCO3⫺ causes a net increase in steady-state pHi (78, 121). This elevated pHi is most likely maintained at least in part by NDCBE and NBCn1, which are robustly expressed in pyramidal neurons from this region. In CA1 neurons with a relatively high initial pHi, the switch to CO2/HCO3⫺ has no effect on steadystate pHi or causes it to fall (78, 121). The most straightforward explanation of Church’s data is that he was mainly working with low-pHi neurons. Three studies on genetically altered mice support Church’s hypothesis: the NDCBE- and NBCn2-null mice (which lack a single acid extruder) exhibit signs of reduced neuronal excitability, whereas the AE3-null mouse (which lacks a single acid loader) has a reduced seizure threshold (378). The link between neuronal pHi regulation and excitability is reviewed in References 76, 186, 187, and 898. B) Role in central nervous system plasticity. The switch of glycine evoked responses of cartwheel cells (glycinergic interneurons) in the dorsal cochlear nucleus of mice from excitatory to inhibitory follows the lowering of [Cl⫺]i, which shifts ECl from a value more positive to a value more negative than Vm (see review in Ref. 34). The shift in ECl is

C) Potential contribution to CSF secretion. The basolateral presence of NDCBE protein in the choroid plexus epithelium of fetal, but not adult, rats suggests that NDCBE contributes to CSF secretion in early developmental stages (176). In adult rats, the basolateral step of transepithelial HCO3⫺ transport across the CPE is likely mediated by NBCn2 and, to a lesser extent, by NBCn1. Recall that NDCBE transcripts are present in the choroid plexus of the adult human, where it is possible that NDCBE plays a functional role. III) Urinary system. A) Unproven role in Cl⫺ reabsorption in the PT. Na⫹-dependent Cl-HCO3 exchange has been proposed to contribute to the basolateral step of Cl⫺ reabsorption by the renal proximal tubule (29, 419, 833). However, this hypothesized Na⫹-dependent Cl-HCO3 exchange activity has not been demonstrated to be directly coupled to Na⫹ flux in the proximal tubule, and is difficult to isolate experimentally due to the much larger HCO3⫺ flux mediated by the electrogenic Na/HCO3 cotransporter NBCe1 in the same basolateral membrane (29, 419, 677, 833). Three observations speak directly to the issue of whether NDCBE contributes to proximal-tubule Cl– reabsorption: 1) antiNDCBE immunoreactivity has not been observed in the PT;76 2) although removing peritubular Na⫹ does indeed reduce Cl⫺ efflux across the basolateral membrane of the proximal tubule, removing peritubular Cl⫺ does not lead to a change in intracellular Na⫹ activity; and 3) the 1:1 Cl⫺: HCO3⫺ exchange stoichiometry estimated for the activity (506) is different from the expected 1:2 coupling ratio for NDCBE activity (337, 719). The molecular identity of the transporter(s) responsible for this basolateral Na⫹-dependent Cl-HCO3 exchange phenomenon have yet to be determined.77 NDCBE is also suggested to contribute to NaCl reabsorption in the collecting ducts of Na⫹-depleted mice.

76 Immunohistochemistry using an antibody specific to NDCBE-A and -B does not detect NDCBE protein in the proximal tubule (J. Praetorius, personal communication), although this observation does not preclude the presence of NDCBE-C or -D. 77 The difficulty of resolving NDCBE and NDCBE-like activities are discussed on p. 895.

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I) General. A) pHi regulation. Investigators have proposed that NDCBE-like activity plays a key role in pHi regulation in many mammalian cell types, including pyramidal neurons from the CA1 region of hippocampi (850, 889), aortic endothelial cells (280), fibroblasts (157, 531), cultured vascular smooth muscle cells (459, 775), cultured lens cells (33), esophageal epithelia (973), glomerular mesangial cells (114, 115), thyrocytes (477), intrahepatic bile duct cells (354, 913), lymphocytes (786), a monocyte-lymphoma cell line (541), macrophages (949), and mouse oocytes (275). Note that the presence of NDCBE mRNA or protein is not documented for all of these cells types.

subsequent to cellular acidification which follows rapid spiking events (489). The shift requires CO2/HCO3⫺ and is blocked by H2DIDS (489). Immunohistochemistry reveals that these cells express NDCBE and thus, taken together, these data are consistent with the hypothesis that NDCBE contributes to the manifestation of inhibitory signaling (489). Lowering of [Cl⫺]i by an NDCBE-like activity has also been implicated in the development of inhibitory GABA-evoked responses during central nervous system maturation. A similar role is shared by the NDCBE-like SLC4 homolog ABTS-1 in nematodes.

MARK D. PARKER AND WALTER F. BORON

II) Central nervous system. A) Increased protein abundance in some brain regions in response to metabolic acidosis. At the level of the whole brain, NDCBE protein abundance is unperturbed by metabolic acidosis in rats (553). However, at the regional level, the protein abundance in the hippocampal CA3 region, traditionally a region of lower NDCBE expression than other regions of the hippocampus, is 2.5-fold more abundant in acidotic than control rats (553). NDCBE abundance is also increased in some populations of cortical neurons (553). The presumed increase in acid extrusion in these cells would tend to counter decreases in pHi causes by acidosis. B) Lack of increased protein abundance in response to hypercapnia. Different from the response of NBCn1 in the brain, but similar to the response of NBCn2, NDCBE protein abundance is not increased in the brain of mice exposed to chronic hypercapnia (463). III) Lymphatic and immune systems. A) Increased transcript abundance in a model of systemic lupus erythematosus. Systemic lupus erythematosus (SLE) is an autoimmune disease causing inflammation in multiple organs. Two mutants of the T-cell receptor ␨ chain have been linked to SLE, and overexpression of these unstable mutants in murine T-cells is associated with an eightfold increase in NDCBE transcripts in these cells (983). The cause and effect of this upregulation remains to be studied, although it has been noted that apoptosis of thymocytes, T-cell precursors, is increased by cellular alkalinization, yet is inhibited by stilbene derivatives, consistent with a proapoptotic action of

902

NDCBE (980). Note that this proapoptotic effect of NDCBE contrasts with the antiapoptotic effect of NBCn1 in osteoclasts. IV) Urinary system. A) Increased transcript abundance and activity in a renal cell line in response to metabolic acidosis. In a mouse collecting duct cell line, metabolic acidosis increases NDCBE transcript abundance and induces a robust DIDS-sensitive, Na⫹- and HCO3⫺-dependent pHi recovery attributed to NDCBE. These results are consistent with a protective role for NDCBE during metabolic acidosis (35). B) Increased NDCBE-like activity in CCD of alkali-loaded rabbits. Although the renal CCD normally reabsorbs HCO3⫺, the CCD in alkali-loaded animals secretes HCO3⫺ and thereby tends to restore a normal (i.e., less alkaline) blood pH (636). In alkali-loaded rabbits, the blood-to-lumen movement of HCO3⫺ across the basolateral membrane of CCD ␤-intercalated cells involves a stilbene-sensitive, Na⫹ and Cl⫺-dependent mechanism consistent with the activity of NDCBE (271). However, the molecular identity of the transporter(s) responsible for this activity is presently unknown. C) Increased NDCBE-like activity in the CCD of Na-deficient mice. Feeding mice a Na⫹-restricted diet leads to the upregulation of a novel thiazide-sensitive NaCl reabsorption pathway in cortical collecting ducts, contributing to an increase in Na⫹ reabsorption (959). Leviel and co-workers make three observations consistent with a contribution of NDCBE to the NaCl-reabsorption pathway (571): 1) NDCBE protein is expressed in mouse CCD preparations, 2) an apical thiazide-sensitive NDCBE-like activity is upregulated in the intercalated cells of Na⫹-depleted mice, and 3) the CCDs of NDCBE-null mice that have been fed a Na⫹-restricted diet are unable to reabsorb NaCl (571). According to the authors’ model, the parallel action of apical pendrin would recycle HCO3⫺ out of the cell and mediate the requisite uptake of Cl⫺ (571). Not demonstrated are stilbene sensitivity of the NaCl reabsorption pathway and the presence of NDCBE protein in the apical membranes of CCD intercalated cells. I) CAUSES OF NDCBE DOWNREGULATION. I) Central nervous system. A) Decreased protein abundance in brain in response to hypoxia. Chronic continuous hypoxia (CCH) decreases the amount of NDCBE protein in the cortex, subcortex, hippocampus, and cerebellum of adult rat brains, but generally not in neonates (175). CCH also reduces the abundance of NBCn1 and NBCn2.

II) Reproductive system. A) Decreased transcript abundance in testes of feminized mice. NDCBE transcripts are abundant in testis, but virtually absent in the testes of feminized mice, that is to say, mice with a disrupted androgen receptor, or those whose testes cannot descend due to sur-

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H) CAUSES OF NDCBE UPREGULATION. I) General. A) Increased NDCBE-like activity in response to cell shrinkage. In Chinese hamster ovary cells, a rise in pHi upon exposure to hypertonic medium is dependent on extracellular Na⫹, Cl⫺, and HCO3⫺, consistent with the activity of NDCBE (793). It is unlikely that NHE1 contributes inasmuch as the pHi increase is insensitive to amiloride. A question that arises is whether Na⫹-driven Cl-HCO3 exchange would contribute to a net increase in intracellular osmotically active particles, and thereby to a regulatory volume increase. If the nonHCO3⫺ buffering power of the cell were infinite (so that pHi did not change), then the Na⫹-driven Cl-HCO3 exchanger would be osmotically silent (219): the uptake of 1 Na⫹ would be balanced by the efflux of 1 Cl⫺, and the equivalent uptake of 2 HCO3⫺ would have no net effect as intracellular buffers released H⫹ to titrate the HCO3⫺ to CO2 ⫹ H2O, which would exit the cell. However, at finite non-HCO3⫺ buffering powers, Na⫹-driven Cl-HCO3 exchange activity would cause pHi to rise, leading to a rise in [HCO3⫺]i, more so for lower buffering powers, which would in principle contribute to cell swelling. Finally, to the extent that a rise in pHi stimulates Cl-HCO3 exchange, the net effect would be the intracellular accumulation of NaCl, which would contribute to cell swelling.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

gical intervention prior to puberty (693). The authors suggest that downregulation of this and other transporter activities may perturb the composition of seminiferous fluid, inhibiting germ cell maturation and contributing to the feminized phenotype. J) CONSEQUENCES OF NDCBE DYSFUNCTION.

NDCBE null-mice exhibit reduced neuronal excitability and renal Na⫹-reabsorption defects. Human pathologies that are linked to NDCBE dysfunction have yet to be described.

1) In the pyramidal layer of the CA1 region of hippocampal slices prepared from NDCBE-null mice, the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) is twofold less than in preparations from wild-type mice (889). A reduction in mEPSC frequency can be mimicked in wildtype preparations by simultaneously lowering both pHo and pHi. Moreover, an increase in mEPSC frequency occurs in preparations from NDCBE-null mice with an increase in either pHo and pHi together or pHi alone. These results are consistent with the idea that the reduced excitability in NDCBE-null mice results from a low pHi (889). 2) In the CA1 region, the amplitude of population spikes evoked by stimulating Schaffer collaterals is lower in NDCBE-null than in wild-type mice (889). Wild-type mice subjected to a second round of stimulation exhibited population spikes with a 1.5-fold greater amplitude than in the first round, whereas NDCBE-null mice exhibited population spikes with a 2-fold greater amplitude than in the first round, consistent with a greater presynaptic plasticity in the mutant mice (889). 3) During repetitive stimulation, the time constant for the release of vesicle contents from boutons of hippocampal slices was greater (i.e., the release was slower) in NDCBEnull versus wild-type mice (889). 4) NDCBE-null mice have an increased latency until onset of seizures/ictal activity in response to interperitoneal administration of the proconvulsive substances pentylenetetrazol and pilocarpine and to hyperthermia (889). II) Urinary system. A) Defective regulation of NaCl reabsorption. NDCBE-null mice fed a Na⫹-deficient diet are unable to upregulate a thiazide-sensitive NaCl reabsorption pathway that would tend to enhance Na⫹ retention (see Ref. 571). Thus, under conditions of Na⫹ restriction, NDCBE dysfunction might be expected to be associated with volume depletion.

A) SUMMARY.

The electroneutral Na/HCO3 cotransporter NBCn2 (encoded by the SLC4A10 gene) cotransports Na⫹ and HCO3⫺ with accompanying futile cycles of Cl-Cl exchange. NBCn2 appears to undergo a mode switch into a Na⫹-driven Cl-HCO3 exchanger (“NCBE”) under certain assay conditions when extracellular Cl⫺ is unavailable. Some investigators report that mouse and rat Slc4a10 products act in NCBE mode even under physiological conditions. In common with several other NCBTs, NBCn2 is stimulated by the soluble protein IRBIT. NBCn2 is present in many organs but is notably abundant in the central nervous system, where its action is predicted to enhance neuronal excitability. Such a role is corroborated by a study of NBCn2-null mice. Genetic disruption of the SLC4A10 gene locus in humans is linked with autism and epilepsy. B) NOMENCLATURE OF Slc4a10 PRODUCTS.

Slc4a10 products were initially termed NCBE following a report that mouse Slc4a10 functions as a Na⫹-driven chloride/bicarbonate exchanger in both Xenopus oocytes and HEK-293 cells (1021). However, under near-physiological conditions, the isotopic Cl⫺ efflux associated with human SLC4A10 activity in Xenopus oocytes does not require extracellular Na⫹ and does not represent a net movement Cl⫺ but rather Cl-Cl exchange. Thus,the human SLC4A10 product normally functions as an electroneutral Na/HCO3 cotransporter (FIGURE 38A) that the authors propose to rename NBCn2 (719), an acronym that we will use in following sections in place of NCBE. C) MOLECULAR ACTION OF NBCN2. Four functional studies demonstrate that NBCn2, whether it is human NBCn2 heterologously expressed in Xenopus oocytes, or mouse or rat NBCn2 in mammalian cells, mediates a Na⫹-dependent HCO3⫺ uptake that can be blocked by DIDS (212, 317, 719, 1021). One of these studies further demonstrates that the

NBCn2

Na+ HCO3– Cl–

Na+ HCO3– HCO3–

A

B

Cl–

Cl–

FIGURE 38. Molecular action of NBCn2. Possible molecular mechanisms by which NBCn2 could operate in an electroneutral ⫺ with accompanying futile cycles mode to cotransport Na⫹ and HCO3 ⫺ of HCO3 -dependent Cl-Cl self-exchange (A). In the absence of extracellular Cl⫺, NBCn2 performs Na⫹-driven Cl-HCO3 exchange (B). The Slc4a10 gene product from mice and rats is reported to act like NDCBE (FIGURE 35) even in the presence of extracellular Cl⫺. Note 2⫺ or that we have not considered any models that are based on CO3 H⫹ cotransport.

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I) Central nervous system. A) Reduced network excitability and increased presynaptic plasticity in NDCBE-null mice. As discussed, the action of NDCBE is hypothesized to enhance neuronal excitability. Four observations demonstrate that neuronal excitability is reduced in NDCBE-null mice.

3. NBCn2/NCBE (Slc4a10)

MARK D. PARKER AND WALTER F. BORON

I) A study of mouse Slc4a10 expressed in Xenopus oocytes. When expressed in Xenopus oocytes, mouse Slc4a10 mediates HCO3⫺-dependent isotopic influxes of Na⫹ and Cl⫺ and efflux of Cl⫺. This Cl⫺ efflux is maximal in the collective presence of extracellular Na⫹, Cl⫺, and HCO3⫺. In their original description of “NCBE,” Wang and co-workers (1021) interpreted these data as evidence for Na⫹-driven Cl-HCO3 exchange. However, three additional observations in the same study are more consistent with Cl-Cl exchange in parallel with Na/HCO3 cotransport rather than a classical model of Na⫹-driven Cl-HCO3 exchange activity. 1) The transporter mediates an influx (in addition to an efflux) of 36Cl in the presence of extracellular Na⫹ and HCO3⫺, 2) Cl⫺ influx does not require extracellular Na⫹ or HCO3⫺, and 3) Cl⫺ efflux requires extracellular Cl⫺ (i.e., trans-side dependence). II) A study of human SLC4A10 expressed in Xenopus oocytes. To address the question of whether the transporter mediates a net efflux of Cl⫺, Parker et al. (719) in a later study expressed human NBCn2-B in oocytes and used a Cl⫺-sensitive microelectrode to monitor [Cl⫺] on the extracellular surface ([Cl⫺]S) of the oocyte. Bulk extracellular [Cl⫺] was maintained at 10 mM to enhance electrode sensitivity. The authors found that, when exposed to CO2/ HCO3⫺, oocytes expressing either AE1, human NDCBE, or squid NDCBE exhibited a transient rise in [Cl⫺]S, indicating a HCO3⫺-stimulated net efflux of Cl⫺. However, oocytes expressing NBCn1 or NBCn2-B exhibited no [Cl⫺]S increase. Thus, under these conditions, SLC4A10 does not mediate a net efflux of Cl⫺. The authors also found that the NBCn2-mediated 36Cl efflux that is stimulated by application of HCO3⫺, is independent of the presence of extracellular Na⫹. Instead, the 36Cl fluxes must represent a futile Cl-Cl exchange that accompanies the true physiological function, the apparent 1:1 coupled influx of Na⫹ and HCO3⫺ (FIGURE 38A).

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Although the preceding study demonstrates that NBCn2 does not normally mediate Na⫹-driven Cl-HCO3 exchange, an interesting observation is that human NBCn2-B appears to be capable of Na⫹-driven Cl-HCO3 exchange under a particular nonphysiological condition, namely, the absence of extracellular Cl⫺. Removing extracellular Cl⫺ reduces 36Cl efflux by half (presumably by eliminating Cl-Cl exchange), and the remaining 36Cl efflux now requires both extracellular Na⫹ and HCO3⫺ (719). Moreover, in the absence of extracellular Cl⫺, NBCn2 mediates a robust, pHi recovery. Thus it appears that, with no extracellular Cl⫺ to participate in Cl-Cl exchange, the transporter is now obligated to engage in Na⫹-driven ClHCO3 exchange (FIGURE 38B). III) Studies of rodent Slc4a10 expressed in mammalian cells. In the case of mouse NBCn2-B heterologously expressed in HEK-293 cells (1021) or mouse NBCn2-A and rat NBCn2C/D heterologously expressed in 3T3 cells (212, 317), removing extracellular Cl⫺ blocks HCO3⫺ influx. The most straightforward explanation for these data is that removing extracellular Cl⫺ switches the activity of Slc4a10 from electroneutral Na/HCO3 cotransport to Na⫹-driven Cl-HCO3 exchange, as predicted by Parker et al. (719), but that the concomitant depletion of intracellular Cl⫺ eliminates this activity. On the other hand, if extracellular Cl⫺ removal fails to deplete intracellular Cl⫺ over the time period examined, then an alternative explanation is that the rodent transporter expressed in mammalian cells behaves differently than the human transporter expressed in oocytes. A study of rodent-Slc4a10-transfected 3T3 cells (212) includes four novel observations that are provided as evidence that Na⫹-driven Cl-HCO3 exchange is the normal mode of action for Slc4a10. 1) Slc4a10-transfected cells alkalinize at a faster rate than nontransfected cells in response to the acute removal of extracellular Cl⫺ as if, in the absence of Cl⫺, the driving force for Cl⫺ efflux and thence Na/HCO3 influx is increased (212). This observation agrees with the findings in Reference 719, namely, that NBCn2 can act as a Na⫹-driven Cl-HCO3 exchanger in the absence of bath Cl⫺. The 3T3cell data are complicated by the presence of substantial endogenous anion exchange activity in these cells (212) that would tend to enhance the rate of alkalinization upon Cl removal in Slc4a10-transfected cells. 2) 36Cl efflux occurs at a greater rate from 3T3 cells transfected with rat Slc4a10 and bathed in a HCO3⫺-buffered solution compared with similar cells bathed in a HEPESbuffered solution, but only in the presence of Na⫹ (212). In this respect, the behavior of rat Slc4a10 in 3T3 cells appears to differ from the behavior of human NBCn2. When expressed in oocytes, human NBCn2-B mediates a Cl⫺ efflux that is independent of the presence of extracellular Na⫹ (719).

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transport mediated by human NBCn2 is electroneutral (719). Three groups provide evidence that the Na⫹ -dependent HCO3⫺ influx mediated by NBCn2 is accompanied by the efflux of 36Cl (212, 719, 1021). However, controversy has arisen over whether this efflux represents a net movement of Cl⫺ under physiological conditions. Two groups of investigators (212, 1021) interpret the Cl⫺ efflux data as evidence that mouse and rat NBCn2 mediate Na⫹-driven Cl-HCO3 exchange (like NDCBE) under physiological conditions (FIGURE 38B). A third group (719) provides evidence that the 36Cl efflux that accompanies human NBCn2 action represents futile cycles of Cl-Cl exchange under physiological conditions (FIGURE 38A) and that NBCn2 is a second electroneutral Na/HCO3 cotransporter (the other being NBCn1). However, NBCn2 does behave as a Na⫹-driven Cl-HCO3 exchanger in the absence of extracellular Cl⫺ (719). The evidence pertaining to NBCn2-mediated Cl⫺ movement in these studies is considered below.

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

3) Cl⫺ efflux, but not Cl⫺ influx, is enhanced in the presence of HCO3⫺, an observation offered as evidence that the Cl⫺ efflux mediated by mouse Slc4a10 represents a net efflux (212). An alternative explanation is that the extent of 36Cl influx over the 2-min duration of the influx assay is underestimated due to the simultaneous 36Cl efflux mediated by NBCn2. With human NBCn2-B expressed in oocytes, 36Cl efflux also is enhanced by HCO3⫺, which simply appears to stimulate Cl-Cl exchange (719).

In summary, it is unknown whether human SLC4A10 and rodent Slc4a10 exhibit true functional differences, or whether the disparities between the studies are methodological in nature. Indeed, as human SLC4A10 is capable of shifting between “NCBE” and “NBCn” modes, it is not inconceivable that rodent Slc4a10 could behave differently from human SLC4A10. D) THE Slc4a10 GENE.

The human SLC4A10 gene occupies 27 exons over ⬃360 kb (FIGURE 39, A and B) on chromosome 2q24.2 (1081). A singleton EST (GenBank DNA accession no. BP229748) from fetal brain provides evidence that the SLC4A10 locus may extend 150 kb further upstream than presently thought, filling the apparent gap between SLC4A10 and its upstream neighbor TBR1. TBR1 encodes a transcription factor that is expressed in cortical neurons during development (133). The TBR1 paralog EOMES/TBR2 is the upstream neighbor of SLC4A7 (FIGURE 31A), indicating a longstanding relationship between these two gene families. The downstream neighbor of SLC4A10, DPP4, encodes the plasminogen receptor dipeptidyl peptidase IV (328). DPP4 protein interacts with and enhances the activity of NHE3 in the proximal tubule (324, 325) and also dampens stimulation of duodenal HCO3⫺ secretion by degrading glucagon-like peptide (418). It is unknown whether DPP4 influences the activity of NCBTs. Intron 1 of SLC4A10 contains a binding site for the transcription factor and tumor-suppressor p53 (1031). SLC4A10 transcription is downregulated in a colon carcinoma cell line by 5-fluorouracil induction of p53 expression (see supplemental data for Ref. 1031). It would be interesting to

E) STRUCTURAL FEATURES AND VARIANTS OF NBCn2.

Variation among mammalian NBCn2 transcripts arises by alternative splicing at any or all of the following three sites in Slc4a10 pre-mRNA (317). I) Sources of variation in coding sequence among NBCn2 variants. The NBCn2 gene is only known to include a single promoter. As depicted in FIGURE 39C and FIGURE 40, there are two major sources of variation between NBCn2 transcripts: the optional inclusion of a 30-amino acid cassette in the Nt and the choice of one of two Ct appendages (a 22-amino acid appendage that ends “-ETCL” or a 4-amino acid appendage that ends “-SSPS”). A further, minor source of variation arises due to the optional inclusion of a single alanine residue due to an apparently degenerate splice boundary. All four NBCn2 clones reported to date are predicted to include an autoinhibitory domain and IRBIT binding determinants in their Nt (718, 722).

A) Optional Ala. The 5= end of exon 7 which, at least in rat NBCn2, contains a cryptic splice-site that, when utilized by the splice machinery, shortens the transcript by three nts (CAG) resulting in the loss of a single Ala residue from the Nt of the transporter. Thus the Ala is optional in rats. In humans, the Ala is always present. B) Cassette A. NBCn2 transcripts can differ in the inclusion or exclusion of exon 8, sometimes referred to as DNA cassette or insert “A,” the excision of which by splicing removes sequence that encodes a 30-amino acid protein “cassette A” (FIGURES 39C and 40). The inclusion of cassette A is predicted to extend the Nt loop (FIGURE 15). C) Choice of alternative Ct (“-SSPS” or “-ETCL”). Exon 26, sometimes referred to as DNA cassette or insert “B,” encodes the 4-amino acid Ct appendage “-SSPS” (FIGURES 39C and 40). The excision by splicing of cassette B allows translational read-through to an alternative downstream termination codon in exon 27. Thus removal of cassette B produces NBCn2 variants with a longer and different Ct (21-amino acid of NBCn2-C/D replaces 4-amino acid of NBCn2-A/B). The 21-amino acid Ct terminates with a type I consensus PDZ-domain binding motif “ETCL” (317). In an astrocytic cell line, the inclusion of the 21-amino acid Ct appendage in rat NBCn2 (rb2NCBE, see below) results in increased colocalization of NBCn2 with the actin cytoskeleton, compared with rb1NCBE, a variant with the 4-amino acid Ct appendage (317). The cytoskeletal attachment of the 21-amino acid appendage is mediated via EBP50 and ezrin (562). Consistent with the hypothesis that an enhanced cytoskeletal interaction is im-

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4) A comparison of the rates of Na⫹ and HCO3⫺ influx into Slc4a10-transfected cells, calculated from measurements of fluorometric dyes, suggest that the Na:HCO3 cotransport ratio is 1:2 for rodent Slc4a10. Thus the Cl⫺ efflux would have to be net to maintain electroneutrality (212). However, there are potential risks in comparing rates obtained by two different methods (Na⫹- versus pH-sensitive dyes) as well as concerns that the Na dye (CoroNa) might not be suitable for quantitative measurements at low [Na⫹] (see Ref. 641). Cited as validation for the Slc4a10 stoichiometry measurements, the stoichiometry of NBCn1 calculated by this method was, as expected, 1:1.

know whether p53 in fact decreases the expression of NBCn2.

MARK D. PARKER AND WALTER F. BORON

A

Locus 12q13 20 kb

SLC4A10

DPP4

TBR1

B

Gene structure

P

10 kb

1

C

27

8

Transcript variation

M

NBCn2-A

1

7

1

7

1

7

1

7

1

7

9

23

24

25

* 26

27

9

23

24

25

* 26

27

9

23

24

25

* 27

8

9

23

24

25

* 27

8

9

23

M

NBCn2-B

8

M

NBCn2-C M

NBCn2-D M

rb3NCBE

* 27

FIGURE 39. SLC4A10 gene structure and NBCn2 transcript variants. Scale diagrams showing the human SLC4A10 gene locus together with the position of neighboring genes (A), the position of the promoters (P), and the position of exons within SLC4A10 (B). Transcript variants are represented, not to scale, as numbered boxes joined by a horizontal line (C). Each numbered box represents the inclusion of that exon in the mature transcript. “//” denotes that all four transcripts include exons 1–7 and 9 –23. Exons that include the initiator ATG codon (“M”) and termination codon (“*”) are marked for each transcript. Colored exons, or parts of exons, correspond to the protein regions that each encodes, which are identically colored in FIGURE 40. Uncolored exons, or parts of exons, denote untranslated 5= and 3= sequence. Exons that are connected with a dashed line are predicted, but not demonstrated, to be included in the mRNA. Note that rb3NCBE has only been isolated from rat cDNA.

portant for efficient trafficking of the transporter to the plasma membrane, cytoskeletal disruption by cytochalasin B treatment of fibroblasts expressing rb2NCBE results in the loss of transporter activity from the plasma membrane (562). Indeed, in a mouse fibroblast cell line, rb2NCBE variant traffics more efficiently to the plasma membrane than rb1NCBE (317). However, the opposite is observed when NBCn2 variants are expressed in MDCK cells (756) perhaps, the authors suggest, due to the lack of an accessory protein. II) Cloned NBCn2 variants that are demonstrated or likely to exhibit NCBT activity. GenBank protein accession numbers for the variants discussed in this section are provided in Appendix IV. A) NBCn2-A (NCBT activity demonstrated). NBCn2-A lacks the 30-amino acid cassette A and includes the 4-amino

906

acid Ct appendage that ends with “-SSPS.” It is orthologous to the rat variant rb5NCBE. Full-length NBCn2-A transcripts have been isolated from a mouse pancreatic cell line cDNA library (1021), and from rat hippocampus and mouse brain cDNA preparations. In the brains of mice, NBCn2-A appears to be the most abundant NBCn2 variant in the subcortex (598). B) NBCn2-B (NCBT activity demonstrated). NBCn2-B includes the 30-amino acid cassette A and the 4-amino acid Ct appendage that ends with “-SSPS.” It is most orthologous to the rat variant rb1NCBE, which does not include the optional Ala that is always present in humans. Full-length NBCn2-B transcripts have been cloned from human kidney cDNA (719) and from rat hippocampus and mouse brain cDNA preparations. In the brains of mice, NBCn2-B appears to be the most abundant NBCn2 variant in the medulla (598).

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P

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

Ct

C as se tte

100 aa

1–5

6–9

10–14

C (e ass xo e n tte 26 B )

TMD

A

Nt

1,088

NBCn2-A 3

1,118

NBCn2-B 30

3 PDZ

NBCn2-C

1,106

21

30

1,136

21

rb3NCBE

1,054 30

1 (exon 27)

FIGURE 40. NBCn2 protein variants. Scale diagram of protein variants that are encoded by the transcripts represented in FIGURE 39C. Horizontal bars represent protein sequence laid out from Nt to Ct. Vertical bars represent position of ␣-helical TMs. Protein cassettes are labeled with a number denoting their size in amino acids and colored to denote their genetic origin as shown in FIGURE 39C. All NBCn2 variants are presumed to include an autoinhibitory domain and IRBIT-binding determinants in their Nt. A color-matched protein sequence alignment of the variants is provided in Appendix V.

C) NBCn2-C (NCBT activity untested). NBCn2-C lacks the 30-amino acid Nt cassette A and includes the 21-amino acid Ct appendage that ends with “-ETCL.” The orthologous rat variant is rb4NCBE. Full-length NBCn2-C transcripts have been cloned from mouse brain cDNA preparations (598). D) NBCn2-D (NCBT activity demonstrated). NBCn2-D includes the 30-amino acid Nt cassette A and the 21-amino acid Ct appendage that ends with “-ETCL.” The most similar rat variant is rb2NCBE, which does not include the optional Ala that is always present in humans. Full-length NBCn2-D transcripts have been cloned from mouse brain cDNA (598). E) rb3NCBE (NCBT activity untested). This variant cloned from rat brain is similar to NBCn2-D except that, instead of lacking exon 26, it lacks exons 24 –26. The now out-offrame exon 27 encodes a singleton His residue that is immediately followed by a termination codon. Thus, in rb3NCBE, the most Ct 83 amino acids of NBCn2-D are replaced by a single His. By comparison with an artificially truncated version of human NDCBE that has a Ct of similar length (717), rb3NCBE ought to be functional. III) Predicted NBCn2 variants. A) Predicted variants with an alternative Ct “-RS.” Although not yet demonstrated to be included in a full-length transcript, a novel fragment amplified from human brain cDNA includes a partial, outof-frame exon 26 created by the utilization of a cryptic

splice site with exon 26 (see supplemental material of Ref. 719). The internally spliced exon 26 includes the third base position of a codon hung-over from exon 25 (the triplet in the novel fragment now encodes an Arg rather than the usual Ser) and a singleton Ser codon followed by a termination codon. Thus the fragment is predicted to be part of a transcript that encodes a novel variant that terminates in a protein kinase C consensus phosphorylation site “KRS.” Such variants would be identical to NBCn2-A/B except that, in the novel variants, the 2-amino acid “-RS” replaces the 4-amino acid “-SSPS” in NBCn2-A/B. IV) Other NBCn2 variants. A) An unusual variant that represents only the isolated Nt. One variant, rb7NCBE (GenBank DNA accession no. AY579377), which originates from rat brain, is identical to rb5NCBE at the transcript level save for the inclusion in rb7NCBE of sequence derived from a cryptic exon between exons 11 and 12. The novel sequence encodes 18 amino acids followed by a termination codon. Thus rb7NCBE encodes an isolated but near-complete cytoplasmic Nt (equivalent to residues 1– 451 of human NBCn2-A) plus 18 novel residues. It is possible that the premature termination codon included in this mRNA would make it a target for nonsense-mediated decay (170). rb7NCBE is reminiscent of isolated Nt variants of NBCn1 and NDCBE. B) rb6NCBE (potentially legitimate transcript, NCBT activity unlikely). This variant (GenBank DNA accession

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PDZ

NBCn2-D

MARK D. PARKER AND WALTER F. BORON As far as astrocytes are concerned, one group detected NBCn2 transcripts in mouse cortical astrocytes (317), whereas another did not detect NBCn2 protein in rat hippocampal astrocytes (177). The difference may be explained by 1) phenotypic differences between astrocytes isolated from different brain regions (56), 2) astrocytes not maintaining substantial levels of NBCn2 protein despite the presence of mRNA, or 3) a species difference. As to the splice variants expressed, an analysis of transcripts indicates that astrocytes, in distinction to the neurons discussed above, lack NBCn2-A and NBCn2-B messages, but instead are enriched in NBCn2-D transcripts (317).

F) DISTRIBUTION OF NBCn2. NBCn2 is predominantly expressed in the central nervous system. The distribution of NBCn2 in the CNS and in other organ systems is discussed below and compared with the distribution of other NCBTs in TABLE 5.

B) Choroid plexus and dura mater. In immunohistochemistry studies, the most striking anti-NBCn2 immunoreactivity is in the choroid plexus (see cartoon in FIGURE 28). Antibodies raised against the Nt common to all NBCn2 variants, or to one or the other alternative NBCn2 Ct78 (i.e., short Ct versus long PDZ-binding-motif containing Ct) all stain the basolateral membrane of the choroid plexus epithelium in human, mouse, and rat (113, 177, 216, 429, 755, 756). Immunogold staining of mouse choroid plexus epithelial cells from the third and fourth ventricles, using an anti-Ct NBCn2 antibody, confirms the basolateral distribution and shows the protein to be especially abundant in “highly folded membrane processes between neighboring epithelial cells.” This study also confirms the cytosolic disposition of the Ct (755). An analysis of rodent cDNAs indicates that NBCn2-A may be the predominant transcript in choroid plexus (755). NBCn2 transcripts are also present in the dura mater (399).

I) Central nervous system. A) Brain. NBCn2 transcripts are most abundant in and widely distributed throughout the CNS (214, 317, 399, 428, 684, 719, 1021) in the forebrain (frontal, temporal and occipital lobes and olfactory bulb), the limbic system (in the hypothalamus, geniculate nucleus, thalamic eminence, hippocampus, substantia nigra and in the amygdala, caudate nucleus, and putamen of the corpus striatum), and the hindbrain (cerebellum, medulla, spinal cord). In mouse brains, an antibody directed against an epitope common to all known variants of NBCn2 has the highest level of immunoreactivity in the cerebral cortex, cerebellum and hippocampus, and the least in subcortex (174). This pattern is similar to the distribution of NBCn1 but different from that of NDCBE, which is most abundant in the subcortex compared with the other three tested regions (175). Within CA1-CA3 regions of the hippocampus, NBCn2 also exhibits an expression pattern complementary to that of NDCBE, inasmuch as NBCn2 expression is greatest in the CA3 region (429), whereas NDCBE expression appears to be strongest in the CA1 and CA2 regions (176). A study of the developmental expression of NBCn2 transcripts in rodent brains is presented in References 317 and 399. The abundance of NBCn2-A through -D appears to vary among brain regions in mice (317, 598, 600). At the cellular level, NBCn2-A and NBCn2-B transcripts are more abundant than NBCn2-C and NBCn2-D transcripts in neurons (317). In prenatal rat hippocampal neurons, NBCn2 protein is detected by immunocytochemistry in the soma of freshly dissociated cells (177), as well as in the processes (177) and the plasma membrane of the soma (177, 199) of cultured cells (see cartoon in FIGURE 24A).

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II) Sensory organs. A) Eye. In the retinas of mice, NBCn2 transcripts have been detected in the neuronal cell layer and pigment epithelium (399). An NDCBE-like activity, which could be mediated by NBCn2, has been reported in mammalian lens cells (33, 265). B) Ear. NBCn2 transcripts have been detected in the cochlear ganglion (399). III) Peripheral nervous system. A) We are not aware of any reports of NBCn2 expression in the peripheral nervous system. IV) Respiratory system. A) Lung. A mutation in the SLC4A10 gene is associated with lung cancer, although the expression of NBCn2 in healthy or cancerous lung tissue has not been formally demonstrated.

78 The antibody raised against the short Ct of NBCn2-A/B has been shown to cross-react with the long Ct of NBCn2-C/D (756), although RT-PCR results predict that only NBCn2 variants with a short Ct are expressed in mouse choroid plexus (755).

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no.AY579376), which originates from rat brain cDNA, is identical to rb5NCBE at the transcript level (i.e., like NBCn2-A, it omits cassette A and includes cassette B) except for the alternative splicing, at nonconsensus splice sites, of exons 14 and 15. The effect is that the last twothirds of exon 14 are discarded, together with the first half of exon 15. Furthermore, the remaining exon 15 sequence is out of frame and encodes only seven amino acids followed by a termination codon. The resulting rb6NCBE protein product encodes the entire Nt and TM1–3 of NBCn2. However, the frame shift and premature termination at a point within putative TM4 make it unlikely that this product is functional or even stable.

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

V) Circulatory system. A) Heart. NBCn2 transcripts have been detected in preparations of heart ventricles from mice (31). VI) Musculoskeletal system. A) Skeletal muscle. NBCn2 transcripts have been detected in human skeletal muscle preparations (see supplemental data in Ref. 719). Transcripts including the PDZ-binding domain (i.e., NBCn2-C and -D) appear to predominate over those lacking the PDZ binding domain (i.e., NBCn2-A and -B; see supplemental data of Ref. 719). VII) Upper digestive system. A) Stomach. NBCn2 transcripts have been detected in preparations of human (214) and mouse (399) stomach.

IX) Lymphatic and immune systems. A) Lymph node and spleen. According to an NCBI-curated database of ESTs, the human lymph node is a potential site of NBCn2 transcription (Appendix VI). NBCn2 transcripts have also been detected in preparation of rat spleen (317). X) Endocrine system. A) Pancreas. NBCn2-A was originally cloned from a mouse pancreatic beta-cell line (1021). B) Pituitary gland. NBCn2 transcripts have been detected in a preparation of rat pituitary glands (1021). XI) Urinary system. A) Kidney. The archetypal NBCn2-B variant was cloned from human kidney cDNA (719), and transcripts have also been detected in rat kidney preparations (1021). In humans, NBCn2 transcripts are present at least in the renal cortex (214). XII) Reproductive system. A) Male. NBCn2 transcripts have been detected in preparation of rat and mouse testes (1021) and in preparations of mouse epididymis and vas deferens (599). B) Female. NBCn2 transcripts have been detected in preparations of ovary, uterus, and vagina of mice (599). G) PHYSIOLOGICAL ROLES OF NBCn2.

At present, studies of NBCn2-specific activity in situ are few in number. The major issues, to some extent common to all the NCBTs, are that 1) a cell (particularly neurons and choroid plexus epithelia) may express more than one NCBT, 2) specific blockers are not available, 3) physiological dissections of NCBTs are not straightforward because of the difficulty of performing a sufficiently wide range of assays on one cell, and

I) Central nervous system. A) Neuronal excitability. A comparison of average resting pHi values of cells in mouse brain slices shows no significant different between wild-type and NBCn2-knockout mice (429). Even so, the knockout of NBCn2 substantially slows the HCO3⫺-dependent pHi recovery from an intracellular acid load in a mouse brain slice from the hippocampal CA3 region and isolated cells from the mouse choroid plexus (429). As discussed earlier in this review, a faster recovery of pHi following neuronal firing leads to a faster recovery of neuronal excitability. B) CSF secretion. Basolateral NBCn2, along with other basolateral NCBTs (FIGURE 28), is suitably positioned to mediate the basolateral step in the transepithelial movement of Na⫹ and HCO3⫺ from the blood into the CSF, thereby contributing to CSF secretion. This role appears to be confirmed by exhibition of CSF secretion defects in an NBCn2null mouse, although other transporters are perturbed in the CPE of these mice. C) Possible role in central nervous system maturation. The detection of NBCn2 transcripts in the CNS of embryonic mice led to the hypothesis that the expression of Slc4a10 is a developmental switch in which the gene-product lowers [Cl⫺]i and thereby shifts ECl from a value more positive to a value more negative than Vm. Such a shift in ECl would causes the GABA-evoked response to change from excitatory to inhibitory (399). Indeed, the probable Na⫹-driven Cl-HCO3 exchanger ABTS-1 fulfills this role in nematodes. However, the underlying premise of this hypothesis in mice is that Slc4a10 mediates Na⫹-driven Cl-HCO3 exchange. Inasmuch as human NBCn2 is unable to effect net Cl⫺ movements under physiological conditions (719), the original hypothesis is unlikely to be correct in humans. NDCBE action could theoretically fulfill this role in humans, given the correct temporal expression pattern. II) Reproductive system. A) Possible role in sperm capacitation. In 1996, Zeng et al. (1090) reported that the recovery of pHi in sperm following an acid-load is stilbene-sensitive and requires Na⫹, Cl⫺, and HCO3⫺ (1090). Subsequently, Wang et al. (1021) speculated that NBCn2 contributes to the alkalinization of sperm required for their capacitation. This speculation could be correct if 1) the removal of external Cl⫺ converted the activity of NBCn2 to Na⫹-driven Cl-HCO3 exchange, 2) the depletion of intracellular Cl⫺ blocked this Na⫹-driven Cl-HCO3 exchange activity, and 3) the NDCBE-like activity is not mediated by

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VIII) Lower digestive system. A) Widespread. NBCn2 transcripts have been detected in preparations of human duodenum (214), rat ileum (1021), and human liver (719). In the liver, NBCn2-A appears to be more abundant that NBCn2-B-D (see supplemental data of Ref. 719).

4) evidence from knockout mice is complicated by dysregulation of other transporters. Ideally, one might use immunocytochemistry or single-cell PCR to verify that an identifiable cell has a single NCBT, which could be approached with standard techniques for studying pHi regulation. Failing that, knockdown approaches are promising, although one must remain wary of secondary effects.

MARK D. PARKER AND WALTER F. BORON NDCBE. However, formal demonstration of NBCn2 expression in these cells is presently lacking. H) CAUSES OF NBCn2 UPREGULATION.

To our knowledge there are no reports of maneuvers that increase the transcript abundance, protein abundance, or plasma membrane abundance of NBCn2. However, preliminary reports show that the functional expression of NBCn2 is enhanced by coexpression with IRBIT (718, 722).

I) CAUSES OF NBCn2 DOWNREGULATION. We are not aware of any reports of maneuvers that decrease NBCn2 transcript abundance. Two studies have reported maneuvers that downregulate NBCn2 at other levels.

II) Central nervous system. A) Decreased protein abundance in the brain in response to hypoxia. NBCn2 protein levels generally fall in response to chronic continuous hypoxia (CCH) in the hippocampus, cerebral cortex, subcortex, and cerebellum of neonatal and adult mice (174). Downregulation in hypoxia is also characteristic of NBCn1 and NDCBE, except that the downregulation of NDCBE occurs in adults but generally not in neonates (175). B) Lack of decreased protein abundance in response to hypercapnia. Neither NBCn2 nor NDCBE protein abundance is increased in the brains of mice exposed to chronic hypercapnia (463). J) CONSEQUENCES OF NBCn2 DYSREGULATION.

As expected for a gene most abundantly expressed in the central nervous system, most reported signs of NBCn2 ablation in mice and pathologies linked to the SLC4A10 gene in humans relate to the brain and bear on changes in neuronal excitability (e.g., reduced sensitivity to proconvulsants, epilepsy, autism). Defective CSF secretion is also described in NBCn2-null mice, but the molecular basis of this pathology appears to be complex and may not be primarily due to loss of NBCn2 activity per se. A single report of a genetic linkage between SLC4A10 and lung cancer bears on the consequence of NBCn2 dysfunction outside of the brain.

I) General. A) Potential role in tumor growth. A report that Na⫹-dependent Cl-HCO3 exchange activity attributed to NBCn2 is important for pHi regulation, and therefore proliferation, in the breast cancer cell lines EMT6, MCF7, and MDA-MB231 (1041), must be interpreted with caution.

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II) Central nervous system. A) Reduced neuronal excitability in mice with a disrupted Slc4a10 gene. As noted above, in the hippocampal CA3 region, the recovery of pHi from an acid load is slower with an NBCn2-null than with a WT mouse. Moreover, in both NBCn2-null and WT mice, the frequency of 4-aminopyridine–induced seizure-like events in the CA3 region is reduced by neuronal acidification (429). Thus it is not surprising that the subsequent recovery in the frequency of these seizure-like events is slower in the knockout than in the WT mice (429). Consistent with this indication of reduced neuronal excitability, NBCn2-knockout mice have an increased tolerance to seizure induction, both in terms of latency until onset and in survival rate (429). B) Genetic linkage to epilepsy in humans. Gurnett et al. (360) described a 13-year-old girl presenting with cognitive dysfunction and complex partial epilepsy was determined to have a balanced chromosomal translocation t(2;13)(q24; q31) involving the SLC4A10 gene. The break point on chromosome 2q24 disrupted SLC4A10 at a point between exons 2 and 3, with the rest of the gene joined at a breakpoint in a gene desert on chromosome 13q31.79 If this translocation event resulted in reduced NBCn2 activity due to haploinsufficiency, then the neurological phenotype would be inconsistent with the described phenotype of Slc4a10 knockout mice, which have no behavioral abnormalities and a reduced, rather than increased, neuronal excitability (429). We consider five possible explanations for this apparent disparity: 1) Systematic difference. A haploinsufficient human is not a null mouse and chemically induced seizures are not complex partial epilepsy. 2) Creation of an uninhibited NBCn2. By real-time quantitative PCR (qPCR), Gurnett et al. (360) found that the level of NBCn2 mRNA (specifically that encoded by exons 1–3) was only about half of normal in lymphocytes from the patient. Thus, although loss of NBCn2 protein could contribute to the disease phenotype, protein levels remain untested in this patient. In addition, it is similarly unknown whether the relocated, telomeric end of SLC4A10 (i.e., exon 3 onwards), from its new locus, might be capable of

79 Build 37.1 of the human genome indicates that the FISH probes used in this study to identify the break point in chromosome 13 in fact bind to 13q22, rather than to the originally assigned 13q31.

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I) General. A) Inhibition of NBCn2 activity by PKA. As expressed in 3T3 cells, NBCn2 activity is regulated by phosphorylation, such that 1) the action of PKA is inhibitory to the functional expression of the transporter and 2) inhibition of PKA enhances functional expression of the transporter (562). It is unknown whether this phenomenon reflects a direct effect of PKA action on NBCn2.

The authors assumed that NBCn2 was the only HCO3⫺ transporter that could mediate recovery of pHi from an acid load in a mammalian cell. In fact, any of the five NCBTs could mediate such a pHi recovery. Subsequent studies have identified NBCn1 as a major pH regulator in MCF7 cells (546), although the presence of NBCn2 in these cells and in other breast cancer cell lines cannot be discounted.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

producing a truncated or alternative gene-product with enhanced function. It is noteworthy that Xenopus oocytes expressing an Nt truncated NBCn2 (i.e., lacking sequence encoded by exons 1–3) exhibit a pHi recovery rate twofold greater than cells expressing the full-length transporter (718). However, the only start codon identified in the SLC4A10 gene to date is that in exon 1.

4) Position effects. The chromosomal translocation might affect the transcription of genes other than SLC4A10, or of microRNAs. Although the report by Gurnett et al. focuses on the SLC4A10 locus on 2q24.2, it is worth noting that 13q22–13q31 is also a susceptibility locus associated with seizures (376). The translocation of the broken arm of chromosome 13 to chromosome 2, and vice versa, might result in altered expression of chromosome 2 or 13-translocated genes such as SCN1A. SCN1A is located at 2q24.3, encodes a voltage-gated Na⫹ channel, and is implicated in epilepsy (669). 5) The phenotype is a function of reduced inhibitory signaling. The loss of NBCn2 could result in a reduced excitability of inhibitory neurons, due to an inability to regulate pHi. Alternatively, if the loss of NBCn2, which can act in an NDCBE-like manner under some conditions, were either directly or indirectly to result in a rise in neuronal [Cl⫺]i, the result might be to convert glycine and GABA signals from the usual inhibitory, to excitatory postsynaptic potentials. Two other epileptic individuals have since been identified as having an SLC4A10 haploinsufficiency. The first individual has a de novo chromosomal deletion of 6.6 Mb that encompasses SLC4A10 and numerous downstream genes in 2q24.2– 2q24.3, including SCN1A, and is both epileptic and mentally retarded (516). The deletion of epilepsy-associated SCN genes in this individual confounds attempts to assess the contribution of NBCn2 loss to this pathology. The second individual has a de novo chromosomal deletion of 6.4 Mb that encompasses SLC4A10 and numerous upstream genes in 2q24.1– 2q24.2. This individual is epileptic, autistic, and mentally retarded (516). Again, the deletion of other genes in this individ-

C) Genetic linkage to autism. A spontaneous deletion of exon 1 of the SLC4A10 gene has been identified in a pair of autistic twins (857). What is presently unclear is 1) whether the two phenomena are linked, 2) how perturbation of the SLC4A10 gene might result in autism, and 3) whether the deletion of exon 1 would result in a haploinsufficiency of functional NBCn2. As noted above, exon 1 includes the only reported initiation codon for NBCn2. It is interesting to note that the twins have not presented with epileptic symptoms. A third individual (discussed two paragraphs above) has a de novo chromosomal deletion of 6.4 Mb that encompasses SLC4A10 and numerous upstream genes in 2q24.1–2q24.2 and is autistic, epileptic, and mentally retarded (516). The contribution of NBCn2 loss to the autistic pathology is difficult to assess inasmuch as autism, in some individuals, is associated with genetic deletions in 2q24.1–2q24.2 that do not encroach into the known extent of the SLC4A10 gene locus (679). Another linkage to autism is to be found in the gene that is the upstream neighbor of SLC4A10: a recent study found that the TBR1 gene-product associates with the product of the AUTS2 autism-susceptibility candidate gene (69). D) Defective CSF secretion in mice with a disrupted Slc4a10 gene. Mice with a targeted disruption of Slc4a10 exhibit a 78% decrease in brain ventricular volume (429). A subsequent study indicates that, in NBCn2-null mice, the deficit of basolateral Na/base cotransport is at least partly compensated by the relocation of normally apical NHE1 to the basolateral membrane (216). Apical NHE1 is in turn replaced by an as yet unidentified amiloride-insensitive NHE. Further preliminary work by Damkier and Praetorius indicates that AQP1 and the Na pump, transporters critical to CSF secretion, also have reduced abundance in the choroid plexus of NBCn2-null mice, reflecting a compensation that would favor cell survival (and thus the integrity of the blood-brain barrier) at the expense of CSF secretion (215). Thus, although the CSF secretion defect in NBCn2-null mice is appropriate, given the location and presumed role of NBCn2, the pathogenesis of this phenotype in NBCn2-null mice is likely more complex than the simple deletion of NBCn2. E) Unproven genetic linkage to depression. Although 2 out of 16 SNPs examined in a study of the human SLC4A10 gene locus were initially linked to major depressive disorder, the linkage was found to be not significant upon further statistical analysis (846). III) Sensory organs. A) Suggested genetic linkage to primary open-angle glaucoma. Defective CSF secretion results in an

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3) Overcompensation. In the choroid plexus of mice, the loss of NBCn2 is compensated by the redistribution of NHE1 in its place (216). It is conceivable that a loss of neuronal NBCn2 might be overcompensated in the proband by a more active population of Na⫹ and/or base transport mechanisms as is the case in NHE1-null mice. Although NHE1-null mice might be expected to be less sensitive to seizures because of their compromised neuronal pHi regulation (1082), they in fact exhibit seizures (70) due to an enhanced neuronal excitability, perhaps caused by the observed compensatory increase in the functional expression of Na⫹ channels (355, 1051). The demonstrated ⬃60% decrease of the acid-loading AE3 protein in NHE1null mice (1060) could also contribute towards a higher pHi and thus a lower seizure threshold (378).

ual makes it difficult to assess the contribution of NBCn2 loss to this pathology. Nevertheless, NBCn2 remains a compelling candidate in the pathogenesis of epilepsy.

MARK D. PARKER AND WALTER F. BORON

IV) Respiratory system. A) Genetic linkage to lung cancer. A genetic linkage study found somatic mutations in SLC4A10 in 2 of 11 lung carcinoma samples.80 One is a P690L substitution at the distal end of EL3, close to the extracellular end of TM6. What effect, if any, this alteration might have on NBCn2 function remains untested. The second polymorphism is not predicted to change the NBCn2 protein sequence, being a synonymous change within the codon for K901, a residue located at the intracellular end of TM11. To date, the expression of SLC4A10 products in healthy or cancerous lung tissue has not been demonstrated.

VI. RELATIVES OF NCBTs IN MAMMALS Here we provide a brief overview of the anion exchangers AE1–3; the three Na⫹-independent, electroneutral ClHCO3 exchangers, which are closely related both structurally and functionally to the NCBTs. This brief analysis should assist in the interpretation of material presented above. Interested readers might consult comprehensive AE reviews, such as those by 1) Jennings (436), who provides an excellent evaluation of the physiological studies that first defined the molecular actions of AE1; 2) Alper (22), who provides an extensive survey of current knowledge concerning the structure, function, splice variants, distribution, physiological importance, and pathologies associated with the AEs; and 3) Cordat and Casey (153) and Romero et al. (805), who provide a thorough consideration of the physiological and pathological importance of HCO3⫺ transporters in general, including the AEs and NCBTs of the Slc4 family, as well as the anion exchangers of the Slc26 family.

80 The data were obtained from the Wellcome Trust Sanger Institute Cancer Genome Project website: http://www.sanger.ac.uk/ genetics/CGP/cosmic.

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In the last part of this section, we summarize current knowledge concerning the two most recently described members of the Slc4 family, Slc4a9 and Slc4a11. Slc4a9 is unique inasmuch as it is like the electrogenic NCBTs in structure but was named as if it mediated Cl-HCO3 exchange. Slc4a11 is unique inasmuch as it is reported to have neither AE-like nor NCBT-like activity, but instead retains the borate-transport activity common to fungal and plantal Slc4like products. However, the functions of both Slc4a9 and Slc4a11 remain controversial.

A. Anion Exchangers (AE1–3; Slc4a1–3) 1. Summary The Cl-HCO3 exchangers of the Slc4 family act as acidloaders (HCO3⫺ export mechanisms) and are the basolateral counterparts of the apically distributed Slc26 family of anion exchangers (FIGURE 1). In erythrocytes, the HCO3⫺ fluxes mediated by AE1 contributes towards the Bohr effect. In addition, red cell AE1 acts as a scaffold protein providing a linkage between the membrane and cytoskeleton, contributing towards maintenance of the structural integrity of the circulating cell. In the kidney, AE1 action contributes towards the maintenance of blood pH and supports urinary acidification. Thus AE1-related pathologies include red cell fragility and whole body acidosis. AE2 exhibits the widest distribution of the three AEs and contributes towards pH balance in a variety of cell types. In the kidney, AE2 contributes towards HCO3⫺ reabsorption in the late PT81 and in the TAL. AE3 is expressed in the eye, brain, and heart. AE3 dysfunction is associated with blindness, epilepsy, and cardiac hypertrophy. 2. Nomenclature AE1, AE2, and AE3 are named for their anion exchange function, which physiologically is the one-for-one exchange of Cl⫺ for HCO3⫺. AE1 is the product of the Slc4a1 gene and is the founder member of the family. AE1 is also referred to as “band 3,” being the third largest protein band evident on coommassie-stained gels of red cell membrane preparations. In older literature, AE1 is sometimes referred to as capnophorin (literally “smoke carrier”). AE2 and AE3 are, respectively, the products of the Slc4a2 and Slc4a3 genes. AE4 refers to the Slc4a9 gene product, which is discussed separately below. 3. Molecular action Under physiological conditions, all three AEs perform Na⫹independent, electroneutral Cl-HCO3 exchange (509, 587, 81 A basolateral Cl-HCO3 exchanger, thought to be AE2, also con⫺ reabsorption in the S3 segment of the proxtributes towards HCO3 imal tubule (506, 677). Although AE2 mRNA has been detected in rat proximal tubule preparations (128), robust AE2 immunoreactivity has not been observed in any segment of the proximal tubules of mice, rats, or humans (27, 158, 914).

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increased pressure differential between the CSF and intraocular compartment that may contribute to the development of glaucoma (briefly reviewed in Ref. 595). Because NBCn2-null mice have reduced ventricle volume, it was hypothesized that variations in the SLC4A10 gene locus might be associated with the incidence of glaucoma in humans. However, a genetic linkage study did not establish a link between affected individuals and seven common SNPs in SLC4A10 (595), and no gross ocular phenotype has been reported for Slc4a10-null mice (429). These data alone do not preclude the possibility that defects in Slc4a10 contribute to glaucoma for four reasons: 1) NBCn2 is a major contributor to CSF secretion and thus a link to glaucoma is sensible, 2) seven SNPs are likely a minor sampling of the true genetic variability among human SLC4A10 genes, 3) none of the 7 SNPs tested has been shown to affect the functional expression of NBCn2, and 4) no studies that describe the lack of ocular pathology in Slc4a10-null mice have been reported.

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

Some have suggested, based on studies of red blood cells, that AE1 is involved in the transport of alkali (i.e., Na and Li) and heavy metals (i.e., Cd, Cu, Mb, Pb, and Zn) carried in the form of anionic complexes (18, 303, 304, 323, 610, 886, 974). For example, Na⫹ is suggested to be transported by AE1 in the form of NaCO3⫺, the same substrate predicted by kinetic analysis to be carried by an NCBT from squid axons (99). At present we can only speculate on how the transport mechanisms of AEs and NCBTs are related, although at least NDCBE and NBCn2 are capable of performing anion exchange (i.e., consistent with NaCO3-Cl exchange) under certain conditions and in principle the Na/ HCO3 cotransport mediated by NBCn1, NBCe1, and NBCe2 could be achieved by variations on a NaCO3-HCO3 exchange mechanism. After correction for transporter abundance, all three fulllength mammalian AE products mediate anion exchange at similar rates (301, 551, 862). Among the three, AE2 is uniquely pHi sensitive, becoming increasingly inactive as pH decreases over the range 9 – 6 (401, 905). Multiple groups have also reported variations in the relative efficacy of anion transport inhibitors between pairs of AEs (e.g., see Refs. 551, 862, 905), although conclusions appear to vary between expression systems. Further distinctions between the paralogs become apparent when we consider individual gene variants and their distribution. 4. Genome AE-encoding genes are considerably more compact (⬃14 –20 kb) than NCBTs genes (⬃100 –360 kb), which is

mainly a function of shorter introns and 3=-UTR regions. The human SLC4A1 gene that encodes AE1 covers ⬃20 kb at chromosomal locus 17q21-q22. The SLC4A2 gene that encodes AE2 covers ⬃17 kb at chromosomal locus 7q36.1. The SLC4A3 gene that encodes AE3 covers ⬃14 kb at chromosomal locus 2q36. Only human chromosome 2 carries more than one SLC4 gene (SLC4A3 and SLC4A10). As previously discussed above, genes that encode AEs and NCBTs share many common exon boundaries (FIGURE 7), which indicates their relatedness. On the other hand, some unique exon boundaries are shared only among AEs and are not shared with NCBTs, indicating that the three AEs diverged from a common ancestor after the divergence of the common NCBT ancestor. 5. Structural features and variants AE proteins are predicted to have a similar structure to NCBTs, due to their sequence similarity at the amino acid level (see FIGURES 2 AND 3). AEs and NCBTs both have a large cytosolic Nt, multiple transmembrane spanning segments, and a relatively short Ct. The crystal structure of the AE1 Nt was first described in Reference 1091 and lowresolution structural reconstructions of the TMD have been reported in References 1022, 1023, 1068, and 1069. One group has suggested that the structure of the AE1 TMD may be similar to that of prokaryotic ClC Cl/H antiporters (1069). There are some key structural differences between AEs and NCBTs, among AEs and among variants of each AE. A) AMINO TERMINUS.

In contrast to the Nt of NBCe1, which is absolutely required for Na/HCO3 cotransport activity (276, 634), the Nt of AE1 is not at all required for basal anion exchanger function (351, 353, 468, 568). However, the Nt of AE1 contains important trafficking determinants (975) and has many protein binding partners (recently reviewed in Ref. 141). The Nt of AE2 is not required for anion exchange activity (587, 1095) but contains determinants that influence the pH sensitivity of AE2 (527, 907, 1095). The necessity of the Nt of AE3 for anion exchange function is untested, but determinants in the Nt of AE3 contribute toward its relatively poor plasma membrane accumulation compared with AE1 and AE2 (301). The three-dimensional structure of the AE1 Nt dimer at pH 4.8 has been solved at 2.6-Å resolution by X-ray crystallography (1091). A subsequent study of the AE1 Nt dimer in solution at neutral and close-to-neutral pH indicates that the original, “low pH” crystal structure is a good representation of the native AE1 Nt structure at physiological pH (1109). B) TRANSMEMBRANE DOMAIN. The AEs have a short third extracellular loop compared with NCBTs. The shortest is that of AE1, which lacks the glycosylation sites that are a common feature of AE2, AE3, and NCBTs. AE1 is also unique in having a glycosylation site in its fourth extracellular loop.

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728), a capability not shared with NCBTs. AEs can also perform futile cycles of HCO3⫺-independent Cl-Cl self-exchange, a mode often exploited in assays as a proxy for physiological AE function (i.e., Cl-HCO3 exchange). Other nonphysiological and minor transport modes described for AEs, but never for NCBTs, include the exchange of monovalent anions such as Br⫺ and NO3⫺ and divalent anions, which are cotransported with H⫹ thereby maintaining electroneutrality, such as SO42⫺ and oxalate2⫺ (e.g., Refs. 359, 401, 434, 439, and 861). In light of these observations, and the observations of Boron and co-workers, which suggest that at least NBCe1 and NDCBE are Na⫹-coupled CO32⫺, as opposed to HCO3⫺, transporters, it is intriguing to speculate that the AEs could be considered to be H⫹-coupled CO32⫺ transporters. The divergence of transporters that perform H⫹ versus Na⫹ coupled cotransport of a particular substrate has been documented for other solute carrier orthologs. For example, members of the Slc23 protein family in mammals are Na⫹ coupled, whereas bacterial Slc23-like orthologs are H⫹ coupled (reviewed in Ref. 940). Due to the permissiveness of the AEs, it is possible that they could be capable of borate transport like their orthologs in plants and fungi. There is some indirect evidence that borohydride (BH4-) is a substrate of AE1 (435).

MARK D. PARKER AND WALTER F. BORON

C) CARBOXY TERMINUS.

The sequence of the Ct is well conserved among AEs and is ⬃40 amino acid in length, far shorter than that of the NCBTs. The AE Ct lacks the characteristic Lys-rich stretches common to all NCBT Cts and the class I PDZ binding motif characteristic of some NCBTs. As is the case with the NBCe1 Ct, the Ct of AE1 contains vital trafficking determinants (184, 203, 300, 976).

D) AE VARIANTS. Each of the three AE genes produces one full-length product and one to three additional truncated variants, transcribed under the control of internal promoters. Thus all AE variants differ only in their extreme Nt sequences (the variants discussed below are depicted in Appendix V). Posttranscriptional processing of AE transcripts is not known to include the splicing that, for NCBTs, results in the optional inclusion of protein cassettes within the Nt and Ct, and variations in extreme Ct sequences.

SLC4A1 contains two alternative promoters. The first produces the full-length gene product erythrocyte AE1 (eAE1; 911 amino acid) and the second produces the truncated kidney AE1 (kAE1; 846 amino acid) that lacks the first 65 amino acid of eAE1 (127, 520, 521) and thereby loses the ability to bind ankyrin (251). A similar transcriptional mechanism produces truncated versions of the AE2 and NDCBE products (e.g., NDCBE-A versus NDCBE-C in FIGURES 36C AND 37). SLC4A2 contains three alternative promoter regions: a, b, and c (1030). The first produce the full-length gene product AE2a (1,241 amino acid in humans, 1,237 amino acid in mice). The second can produces one of two shorter products, AE2b1 or AE2b2, depending on which of two closely positioned transcriptional start sites are utilized. In AE2b1, the first 17 amino acid of AE2a are replaced by a novel 3-amino acid sequence. In AE2b2, the first 17 amino acids of AE2a are replaced by a novel 8-amino acid sequence. In mice, the third promoter region can produce one of two

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even-shorter products, AE2c1 or AE2c2,82 again depending on which of two closely positioned transcriptional start sites are utilized. Mouse AE2c1 is the shortest of all AE2 products and is a truncated version of the other AE2 products, such that AE2c1 initiates at Met199 of AE2a. In mouse AE2c2, the first 193 amino acid of AE2a are replaced by a novel 27-amino acid sequence. The complex transcriptional and posttranscriptional mechanisms that produce each variant are depicted in detail in References 550 and 637. In side-by-side comparisons, AE2b variants have a greater functional expression than AE2a or AE2c1, whereas AE2c2 activity was undetectable (527). It is possible that these observations might at least in part be explained by differences in surface expression; however, AE2c1 clearly has an alkaline shifted pHo dependence (527) compared with the other forms. Alternative promoter choice, resulting in small, seemingly insignificant alterations in Nt sequence, are also common to NBCn1 (e.g., NBCn1-A versus NBCn1-B in FIGURES 31C AND 32). SLC4A3 contains two alternative promoters. The first produces the full-length gene product brain AE3 (bAE3, aka AE3fl; 1,232 amino acids). The second produces the shorter cardiac AE3 (cAE3; 1,034 amino acids) from an alternative transcription site that includes a novel ATG codon. Thus, in cAE3, the first 271 amino acids of bAE3 are replaced by a novel 73-amino acid sequence (588, 1080). One study found that the intrinsic Cl-HCO3 activity of bAE3 is doubled compared with cAE3 and to a truncated AE3 that lacks the unique sequence of both (905), as though the unique longer sequence of bAE3 has a mildly autostimulatory effect. The transcriptional mechanism that produces bAE3 versus cAE3 is similar to that which produces NBCe1-B and NBCe1-A. In fact, the point at which bAE3 and cAE3 sequences converge is only four amino acids downstream of where NBCe1-A and NBCe1-B sequences converge. 6. Distribution Whereas elsewhere we have considered the distribution of NCBTs by organ system, in this section we consider each AE in turn and consider whether its distribution overlaps with that reported for any NCBT. In common with NCBTs, the location of AEs in polarized cells is overwhelmingly basolateral. A) AE1.

eAE1 is prominently expressed in the plasma membrane of red blood cells, whereas kAE1 is located in the basolateral membrane of ␣-intercalated cells in the renal collecting duct (24, 262). Red blood cells have not been demonstrated to express any NCBT. Some researchers describe NBCn1 (694, 1014) and AE4 (498) immunoreactiv82 In rats, AE2c1 and AE2c2 transcripts both encode the AE2c1 polypeptide (1030). Genomic differences may mean that the rabbit and human AE2 genes lack the capacity to produce AE2c transcripts (527).

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As is the case for the NCBTs, glycosylation of AE1 is not essential for transport (155, 352). The AEs lack the four conserved cysteines in EL3 common to NCBTs, although AE2 has a single Cys in this loop. A comparative study of the accessibility of substituted cysteines in the latter half of the TMD indicates structural differences between AE1 and NBCe1 in this region (1112). Lysine-rich motifs, also found in the NCBTS, at the extracellular ends of putative TMs 5 and 13 contribute to the stilbene sensitivity of the AEs (63, 435, 699). Finally, a glutamate in putative TM8 forms an important part of the transport gate of AE1 (437, 438, 440, 443). The three-dimensional structure of the AE1 TMD dimer has been solved at 7.5-Å resolution using cryo-electron microscopy (1068, 1069). However, this resolution is not sufficient to visualize all TMs nor to assign amino acid sequence to regions of electron density.

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

ity in the basolateral membranes of ␣-intercalated cells in the medullary segments of the collecting duct. However, we are unaware of the direct colocalization, even of any two of these three Slc4 proteins, by the same authors in the basolateral membrane of the ␣-intercalated cell.

In many cases the distribution of AE2 overlaps with that of an NCBT. For example, sites of AE2 expression that match those of NBCe1, presented in separate reports, include the basolateral membranes of salivary parotid acinar cells (372, 818), pancreatic acinar cells (816), duodenal enterocytes (25), proximal colon enterocytes (25), late PT, and epididymal epithelia (446). The distribution of AE2 overlaps with that of NBCn1 in the basolateral membrane of mTAL epithelia, and with NBCn2 in the basolateral membrane of choroid plexus epithelia (see cartoon in FIGURE 28 AND Ref. 587). In other cell types, AE2 is coexpressed with NBCe2 but in polar opposite membranes. For example, in choroid plexus epithelia (basolateral AE2, apical NBCe2, see cartoon in FIGURE 28) and in hepatocytes (apical AE2, basolateral NBCe2; see cartoon in FIGURE 29 AND Ref. 46). Reports conflict as to whether NBCe1 is present in ameloblasts, which also express AE2 (see cartoon in FIGURE 20 as well as Refs. 456 and 706). Despite these apparent overlaps in reported distribution, we are not aware of any publications in which the same set of authors has visualized NCBTs and AE2 protein in the same cell. There is no evidence that AEs and NCBTs are capable of forming heterodimers. However, functional coupling of AEs, specifically AE2, and NCBTs has been proposed (571, 1019). For example, NBCn1 in the basolateral membrane of the mTAL presumably mediates the uptake of Na⫹ and HCO3⫺ (571). In parallel, some of the HCO3⫺ exits the cell in exchange for Cl⫺ via AE2. To the extent that the HCO3⫺ fluxes of the two transporters balance, the net effect is NaCl uptake. C) AE3. AE3 is expressed in neurons and glia throughout the central nervous system (378, 463, 502, 509) and in heart preparations (588, 761), specifically in the sarcolemma and T tubules of myocytes (31). Thus the expression of AE3 potentially overlaps with all NCBTs in the CNS and with NBCe1 and NBCn1 in cardiac myocytes. Again, despite the apparently overlapping distribution, NCBTs and AE3 have

7. Physiological roles Here we discuss the generally complementary, but sometimes similar, physiological roles of AEs and NCBTs, and how their different molecular actions impact these roles. A) GENERAL: PHI REGULATION.

AEs typically operate as acidloaders, exchanging intracellular HCO3⫺ for extracellular Cl⫺, tending to restore pHi after an alkaline load (995). NCBTs on the other hand typically operate as acid-extruders, tending, as is the case with Na-H exchangers, to restore pHi after an acid load. The concerted action of these three transport mechanisms over a range of pHi values is nicely demonstrated in a study of ventricular myocytes by Leem and coworkers (563). B) AE1: MAINTENANCE OF RED CELL MORPHOLOGY.

The cytosolic Nt of AE1 forms extensive interactions with cytoskeletal proteins, tethering the red cell cytoskeleton to the red cell membrane. Not only do these interactions help the red cell maintain its biconcave shape, thereby maximizing its surface area-to-volume ratio for gas exchange, but they also allow each cell to be temporarily deformed, rather than sheared, as it passes through the microcirculation. The importance of AE1 for red cell morphology is reviewed by Burton and Bruce (141).

C) AE1: PROMOTION OF GAS EXCHANGE ACROSS THE RED CELL MEMBRANE. Carbon dioxide entering red blood cells in the systemic capillaries is hydrated to H⫹ plus HCO3⫺ via the action of CA II. The HCO3⫺ exits into the plasma via AE1, maintaining a driving force for CO2 entry into the red blood cell and maximizing the CO2-carrying ability of the blood. The H⫹ generated by the CA reaction is buffered by hemoglobin (Hb), which may be tethered to the cytoplasmic Nt domain of AE1 (475). The binding of H⫹ to Hb reduces the affinity of Hb for O2, thereby promoting O2 release (the Bohr effect). The Bohr effect also plays a role in the pulmonary capillaries, where HCO3⫺ entry into the red cell plasma via AE1, maintains a driving force for CO2 exit from the red cell, and promotes H⫹ consumption thereby increasing the affinity of Hb for O2. The relationship between red cell pH and gas exchange is reviewed in Reference 444. ⫺

D) AE1 AND AE2: HCO3 REABSORPTION/H

⫹

SECRETION. In the basolateral membrane of mTAL epithelia (FIGURE 34), AE2 is predicted to move HCO3⫺ from cell to the blood, thereby contributing towards reabsorption of residual HCO3⫺ from the mTAL lumen into the blood (269). HCO3⫺ exit across the basolateral membrane also promotes the generation of intracellular H⫹, which stimulates H⫹ secretion into the lumen. This secreted H⫹ either titrates HCO3⫺ in the lumen (HCO3⫺ reabsorption) or titrates NH3 and other nonHCO3⫺ buffers (H⫹ excretion). Further along the nephron,

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B) AE2. Of the AEs, AE2 is by far the most widely distributed. Locations include the basolateral membranes of epithelia that line the gastrointestinal tract (e.g., see cartoons in FIGURES 21 AND 22 as well as Refs. 25 and 813) and also the basolateral membranes of some renal tubule segments, most prominently in the mTAL (see cartoon in FIGURE 34 as well as Refs. 27, 158, 298, and 914). The AE2a and AE2b variants have a similar broad distribution, whereas robust AE2c expression appears to be restricted to the stomach (25, 550, 1030).

not, to our knowledge, been formally colocalized by immunocytochemistry in the same cell.

MARK D. PARKER AND WALTER F. BORON in the basolateral membrane of collecting duct ␣-intercalated cells, AE1, rather than AE2, performs a similar function (282, 904). The renal actions of AE2 and AE1 both counter metabolic acidosis in the blood. In ameloblasts and osteoclasts, AE2-mediated HCO3⫺ efflux across the basolateral membrane supports H⫹ secretion across the apical membrane, contributing to tooth (456) and bone (1048) remodeling (see FIGURES 20 AND 33). ⫺

E) AE2: HCO3 SECRETION/H

⫹

A consequence of the HCO3⫺ transport function of AEs and NCBTs is that AEs import Cl⫺, whereas NCBTs generally import Na⫹. As noted earlier, both of these consequences can contribute towards the vectoral transport of NaCl, together with osmotically obligated H2O, across epithelia. In airway, duodenal, and colonic epithelia, the functionally coupled action of AE2 and an NCBT (e.g., AE2 and NBCe1 in FIGURE 22) performs, along with NKCC1, Na⫹ and Cl⫺ influx across the basolateral membrane that supports CFTR-mediated Cl⫺ secretion across the apical membrane (312, 397, 1019). Knocking out murine AE2 or NBCe1 is associated with a presumably compensatory increase in NKCC activity in intestinal epithelia (312, 313). F) AE2: SALT SECRETION.

G) AE2: VOLUME REGULATION.

The intracellular alkalinization that follows the shrinkage-induced activation of NHE1 subsequently activates AE2. The resulting net influx of Na⫹ and Cl⫺, followed by water, tends to restore cell volume (449, 873). One group has suggested that shrinkage-induced activation of NDCBE might also tend to restore cell volume. In the red blood cells of trout, swelling opens a cryptic solute channel within AE1 protein (285). The efflux of ions and uncharged solutes through trout AE1 would tend to restore cell volume. Regulated volume decrease is common to the red blood cells of many species, but the physiological involvement of AE1 in such a pathway is not well demonstrated in mammals (357). H) AE3: CONTROL OF NEURONAL EXCITABILITY.

The acid-loading action of AE3 tends to dampen neuronal excitability, con-

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8. Causes of AE upregulation Here as well as in the following section, we mainly consider the effect upon AEs of those perturbations that have been described elsewhere to affect NCBT functional expression. Typically, acid-extruding NCBTs are upregulated by metabolic and respiratory acidosis, the consequence of which is defense of pHi. Even NBCe1, acting as an acid-loader in the proximal tubule, is upregulated by acidosis, increasing HCO3⫺ reabsorption, the consequence of which is defense of plasma pH. ⫺

A) AE1. AE1 plays a role in support of renal HCO3

reabsorption/H secretion in the CCD. Indeed, AE1 transcript and protein abundance increase in response to metabolic acidosis (282, 398, 763, 828, 1002), which is the appropriate response. One study reports that the red blood cells of individuals permanently living at high altitude (e.g., Bolivians) contain 50% more AE1 protein than red blood cells of individuals permanently living at sea level (e.g., Danes, see Ref. 457). ⫹

B) AE2. In the mTAL, AE2 protein abundance is increased in response to metabolic acidosis, a compensatory mechanism that could increase HCO3⫺ reabsorption/H⫹ secretion by this nephron segment (778). AE2 protein abundance in the mTAL is also elevated by NaCl loading, consistent with its role in support of salt secretion (778), as discussed above.

9. Causes of AE downregulation A) AE1. In keeping with its upregulation during acidosis, AE1 protein abundance in the collecting duct is reduced during metabolic alkalosis (828). B) AE3.

AE3 protein abundance is reduced in the brains of rats following a 2-wk exposure to 12% CO2 (463), a response that is consistent with the reduced usefulness of an acid-loading transporter under hypercapnic conditions. 10. Consequences of AE dysfunction Interested readers might refer to the reviews of others for extensive discussions of the pathological consequences of AE1–3 dysfunction (e.g., see reviews in Refs. 22 and 1036). Here we discuss pathologies that are relevant to defects in both AEs and NCBTs and how differences between the transporters may impact the sequelae. A) AE1: DISTAL RENAL TUBULAR ACIDOSIS AND HEMOLYTIC ANEMIA. Individuals with mutations in the SLC4A1 gene (130) and mice with a disrupted Slc4a1 gene (904) have a lower-than-

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REABSORPTION. Although AEs typically support HCO3⫺ reabsorption, in three instances, AE2 is positioned to support HCO3⫺ secretion: 1) AE2 exhibits an apical distribution in cholangiocytes and 2) hepatocytes (46, 987) and 3) AE2 exhibits a lateral distribution in ameloblasts, becoming exposed to the apical compartment during ameloblast maturation by a rearrangement of tight junctions (456). In contrast to AEs, NCBTs typically support HCO3⫺ secretion. Two notable exceptions in the case of NCBTs are NBCe1-A in the basolateral membrane of the proximal tubule and NBCe2 in the apical membrane of the choroid plexus, both of which are predicted to operate with the unusual apparent Na⫹:HCO3⫺ stoichiometry of 1:3, thereby mediating HCO3⫺ reabsorption.

sistent with the association of AE3 dysfunction with epilepsy.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

B) AE2: BONE AND ENAMEL DEFECTS.

Mice and cattle with AE2 insufficiency exhibit signs of osteoclast dysfunction, such as growth retardation and osteopetrosis (432, 455, 643, 1048). On its free or contralacunar membrane surface (facing bone interstitium), the osteoclast expresses AE2 (FIGURE 33). The Cl-HCO3 exchange activity acidifies the cell, thereby promoting H⫹ secretion across the ruffled border by the vacuolar-type H⫹ pumps. As the H⫹ enters the resorption lacuna (the space between the ruffled border and calcified bone), the acidity promotes bone resorption (solubilization of bone mineral and hydrolysis of matrix proteins). Osteoclasts in which NBCn1 abundance has been reduced by antisense technology also exhibit reduced bone-resorption function. NBCn1 appears to be expressed at high levels in the ruffled-border membrane that faces the resorption lacuna, where the action of H⫹ on CaCO3 forms HCO3⫺. Presumably the NBCn1 would move this newly formed HCO3⫺ from resorption lacuna to the cytosol of the osteoclast for removal by AE2 into the interstitium (797). AE2-null mice are toothless, exhibit growth retardation, and die prematurely (314). Mice that are unable to express the a and b variants of AE2 also have defective tooth enamel (126, 616) because AE2 supports H⫹ secretion in ameloblasts. NBCe1 dysfunction is similarly associated with enamel defects, although the underlying mechanism in that case has yet to be resolved.

C) AE2: INFERTILITY. AE2-null mice do not live to breeding age,

but male mice that lack only the a and b variants of AE2 are infertile due to defects in spermiogenesis (638). D) AE2: GASTRIC ACID SECRETION DEFECTS. Gastric secretions in AE2-null mice are not acidic due to a combination of loss of AE2-mediated HCO3⫺ efflux across the basolateral membrane of parietal cells (which normally supports H⫹ secretion), loss of parietal cells, and ultrastructural defects in remaining parietal cells (314). E) AE3: EPILEPSY.

A mutation, A679D, in AE3 reduces the per-molecule activity of the transporter (1004) and is associated with idiopathic generalized epilepsy in humans (830). Furthermore, mice lacking AE3 have a reduced seizure threshold in response to proconvulsive agents (378). The neurons of mice lacking the acid-loading AE3 exhibit an elevated pHi that likely contributes to neuronal hyperexcitability (378). Note that these features of AE3-null mice are opposite to the phenotype of mice that lack the acidextruding transporters NDCBE and NBCn2, which have an increased seizure threshold. F) AE3: BLINDNESS. AE3-null mice exhibit signs of reduced inner retina function and increased apoptosis of photoreceptor cells (30). A similar phenotype is observed in certain strains of NBCe2-null and NBCn1-null mice, the common denominator perhaps being an inability to regulate pHi in these cells, assuming that this is not a side effect of the expression of misfolded protein fragments expressed from the disrupted genes.

B. AE4 (Slc4a9) 1. Summary Despite its reported function as a Cl-HCO3 exchanger in heterologous systems, Slc4a9 is more closely related, at the level of exon-boundary structure and deduced amino acid sequence, to the Na⫹-coupled members of the Slc4 gene family. The molecular action and subcellular distribution of Slc4a9 products remains controversial and may even be species-specific. AE4 expression appears to be mainly restricted to the kidney, most likely in the basolateral membranes of non-␣-type intercalated cells of the collecting duct. As discussed earlier, the Slc4a9 gene seems to have arisen from a recent duplication (the most primordial Slc4a9 known appears in two frog genomes) of an electrogenic NCBT-encoding gene. Among the 10 vertebrate Slc4s, Slc4a9 orthologs have the most divergent sequences. It is possible that this recently duplicated gene is still in the process of diverging. 2. Nomenclature Originally termed hSBC5 (human sodium bicarbonate cotransporter 5) in an early GenBank submission, the

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normal blood pH and [HCO3⫺] and excrete an unusually alkaline urine. Underlying these signs is loss of per-molecule function and/or reduced accumulation of AE1 in the basolateral membrane of collecting duct ␣-intercalated cells (130). The result is an impaired ability of the collecting duct to reabsorb HCO3⫺/secrete H⫹, processes that normally support H⫹ secretion into the duct lumen. Similarly, genetic defects in NBCe1 result in proximal renal tubular acidosis (pRTA), although underlying the acidosis in this case is a failure of HCO3⫺ reabsorption/H⫹ secretion at the level of the PT. Individuals with AE1-associated distal renal tubular acidosis (dRTA) typically have a less severe deficit in plasma [HCO3⫺] because the PT, which is responsible for ⬃80% of the HCO3⫺ reabsorption, is intact. However, patients with an AE1 defect do relatively poorly in acidifying their urine. Individuals with NBCe1-associated pRTA typically have a severe deficit in plasma [HCO3⫺]. Thus the filtered load of HCO3⫺ is low enough that the intact distal nephron can reabsorb the HCO3⫺ and also lower urine pH. (e.g., see Ref. 253). AE1-associated dRTA also have a different set of extrarenal sequelae from NBCe1-associated pRTA because of the different sites of AE1 and NBCe1 expression. For example, dRTA is sometimes accompanied by loss of AE1 from red blood cells, resulting in hemolytic anemia (see review in Ref. 1036) and, secondary to the anemia, cardiac hypertrophy (31, 708).

MARK D. PARKER AND WALTER F. BORON Slc4a9 product was renamed AE4 (anion exchanger 4) following a report that the rabbit Slc4a9 product mediates Cl-HCO3 exchange (982). 3. Molecular action A cDNA encoding an Slc4a9 product was first reported by Tsuganezawa et al. (982). There are three reports of ClHCO3 exchange activity mediated by mammalian AE4.

The authors also reported that Xenopus oocytes expressing rabbit AE4 mediate a Na⫹-independent and DIDS-insensitive 36Cl uptake in the nominal absence of CO2/HCO3⫺. Because they did not examine oocytes in the presence of CO2/HCO3⫺, this result cannot be taken as evidence of ClHCO3 exchange. 2) Ko et al. (498) demonstrate that HEK-293 cells transiently transfected with rat AE4 cDNA alkalinize in response to the removal of bath Cl⫺ in the presence of CO2/ HCO3⫺. This pHi increase appears to be predominantly CO2/HCO3⫺ dependent. The alkalinization is unaffected by lowering of bath Na⫹, but is strongly inhibited by the application of H2DIDS. Evidence of electroneutrality of the transport process is provided by the lack of effect of valinomycin and elevated [K⫹]o on the alkalinization. 3) Xu et al. (1054) expressed mouse AE4 in Xenopus oocytes, using the fluorescence of BCECF to monitor pHi. They reported that, following a 20 –30 min equilibration in CO2/HCO3⫺-containing solution, oocytes expressing AE4 alkalinized upon removal of bath Cl⫺. These authors did not discuss whether pHi recovered from the CO2-induced acid load when the oocytes were exposed to Cl⫺ or whether the alkalinization induced by Cl⫺ removal was DIDS sensitive. An unusual aspect of the data was that the pHi of the control oocyte (i.e., the one not expressing AE4) in CO2/ HCO3⫺ was ⬃7.2 (i.e., roughly the pHi expected in the absence of CO2/HCO3⫺). We would have expected the ap-

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In one preliminary study on Xenopus oocytes, Parker et al. (716) observed that Xenopus oocytes expressing a human AE4 splice variant exhibited a small but significant pHi recovery from a CO2-induced acid load. Moreover, the removal of extracellular Na⫹ produced a very small but significant pHi decrease, consistent with electroneutral Na/ HCO3 cotransport activity (716). We conclude that the functional data on AE4 are inconsistent, possibly due to the use of cDNAs from different species, the use of different heterologous expression systems, different and nonoverlapping protocols, and different experimental approaches. We cannot rule out the possibility that the pHi increases observed after removal of Cl⫺ required a protein endogenous to the host cell. 4. Genome The human SLC4A9 gene occupies at least 22 exons spread over 16 kb at the chromosomal locus 5q31 (591, 720), making it similar in size to the genes encoding AE1, AE2, AE3, and BTR1 but considerably more compact than genes encoding verified NCBTs, which are typically ⱖ100 kb. However, Slc4a9 shares more common exon boundaries with Slc4a4 and Slc4a5, genes that encode electrogenic NCBTs (FIGURE 7). Putative promoter elements are located upstream of the transcriptional starting position of human SLC4A9, including a CCAAT box, a GC box, and a TATA box (720). A region upstream of the first exon of mouse AE4 has basal promoter activity and contains a consensus motif for binding the transcription activator Foxi1. Indeed, AE4 transcription is enhanced 100-fold by Foxi1 (528), and mice lacking Foxi1 also lack AE4 (87) as well as subunits of the H⫹-pump (1003). Sequences with promoter activity are also found within the mouse Slc4a9 gene upstream of exon 3 and upstream of exon 6 (377). 5. Structural features and variants83 A 2001 survey of human ESTs describes 14 distinct AE4 transcripts, most of which are not predicted to encode a functional transporter/stable membrane protein, owing to the presence of stop codons or the absence of transmembrane spans (591). Compared with the most complete reported mammalian transcript, a 15th human transcript (720) lacks part of exon 9 (i.e., leading to the absence of the 11-amino acid “LFGGLIQDVRR” in the cytosolic Nt domain close to TM1) as well as part of exon 12 (i.e., leading to the absence of the 3 amino acid “VSM” in the 3rd extracellular loop). Northern blots of human, mouse, and rabbit

83 GenBank protein accession numbers for the variants discussed in this section are provided in Appendix IV.

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1) Tsuganezawa et al. (982) report that 60% of COS-7 cells transiently transfected with rabbit AE4 cDNA rapidly and reversibly alkalinize in response to the removal of bath Cl⫺ in the presence of CO2/HCO3⫺. The 60% figure is consistent with the 68% transfection efficiency calculated for these cells. Supposed evidence for electroneutrality is provided in experiments in which the authors used a whole cell patch to monitor Vm while applying CO2/HCO3⫺ and then, in the continued presence of CO2/HCO3⫺, removing Na⫹. They observed no substantial Vm changes. It is not clear how this protocol could address the issue of whether the putative Cl-HCO3 exchanger is electroneutral; a better approach would have been to monitor an electrical parameter while removing Cl⫺ in the presence versus the absence of CO2/ HCO3⫺. Only the switch to 130 mM K⫹ produced a Vm change, although the depolarization was slow and poorly reversible.

plication of 5% CO2 to cause pHi to fall to ⬃6.9 and not recover much from there (e.g., see Ref. 725).

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

kidney AE4 RNA demonstrate a multiplicity of bands in these organisms (377, 591, 982). Only two rabbit AE4 variants, AE4a and AE4b, have been cloned, and both have a full complement of transmembrane spans, AE4b differing from AE4a only in the absence of a 16-amino acid sequence within the cytosolic Nt domain of AE4b (982). In mice, Slc4a9 transcription can initiate at multiple points, resulting in the production of Nt truncated AE4 splice variants that are shorter than the longest reported AE4 protein by 157 and 251 amino acids (377). The effect of such truncation on the function and/or trafficking of AE4 remains untested.

E) CIRCULATORY SYSTEM. A study of laser-captured rat brain microvessels demonstrated the presence of AE4 protein by ICAT (isotope-coded affinity tagging) nanoLC-MS/MS (366). F) MUSCULOSKELETAL SYSTEM. We are not aware of any reports of AE4 expression in the musculoskeletal system. G) UPPER DIGESTIVE SYSTEM. I) Salivary gland. AE4 immunoreactivity is reported in the basolateral membranes of duct cells from the mouse submandibular gland (498).

II) Stomach. AE4 transcripts are detected in northern blots of stomach RNA preparations from mouse, rabbit, and rat (498, 1054). Specifically, rabbit AE4 transcripts were detected in preparations from gastric mucous and parietal cells (1054). A 2003 immunohistochemical study reports the presence of AE4 protein in the apical membranes of mouse and rabbit gastric surface mucous cells (1054).

6. Distribution

H) LOWER DIGESTIVE SYSTEM.

AE4 is predominantly expressed in the kidney. The apparent AE4 distribution in specific organ systems is discussed below. Some reports of protein distribution must be regarded with caution due to inadequate characterization of the antibodies used. A) CENTRAL NERVOUS SYSTEM.

AE4 immunoreactivity has been detected in the apical membranes of ciliated ependymal cells in the third ventricle of the choroid plexus of mice and rats (755).

B) SENSORY ORGANS.

I) Intestines. AE4 immunoreactivity is reported in the apical villus membranes of human, rabbit, and mouse duodenum (1054). However, the evidence presented for the presence of AE4 protein in duodenal cells hinges on the specificity of the antibody, which, in mouse preparations, does not immunoreact with a protein of the molecular weight expected for AE4 (see FIGURE 4B of Ref. 1054). Furthermore, others report that AE4 transcripts are absent from mouse duodenum preparations (482, 887). Thus the presence of AE4 in duodenum remains controversial. AE4 transcripts have been reported in rat cecum (498).

As far as we are aware, there are currently no reports of AE4 expression in the eye, ear, or olfactory system.

II) Liver. An NCBI-curated database suggests that the mouse liver is a minor site of AE4 transcription (Appendix VI).

C) PERIPHERAL NERVOUS SYSTEM.

I) ENDOCRINE SYSTEM.

D) RESPIRATORY SYSTEM.

J) LYMPHATIC AND IMMUNE SYSTEMS. We are unaware of any reports of AE4 expression in the lymphatic or immune systems.

As far as we are aware, there are currently no reports of AE4 expression in the peripheral nervous system. AE4 transcripts are detected in cultured human nasal epithelial cells, increasing in abundance as the cells grow to confluence and project cilia (878). The authors of that study report that “immunofluorescent staining was seen along the whole cell membrane, which suggests that AE4 is localized in both the luminal and basolateral membranes.” However, the immunocytochemistry presented in the study, performed on permeabilized cells, does not support the stated conclusion for several reasons. 1) The cells are of undemonstrated confluence and polarity. 2) The confocal microscopic image, which is only in the x-y plane, shows predominantly perinuclear staining, with no evidence of a signal at the plasma membrane. 3) The specificity of the commercial anti-AE4 antibody is not demonstrated.

We are unaware of any reports of AE4 expression in the endocrine system.

K) URINARY SYSTEM.

I) Kidney. Northern blotting studies demonstrate that AE4 transcripts are predominantly renal in humans (591, 720), rats (498), and rabbits (982). In mice, the kidney-specific transcription factor Foxi1 is responsible for the predominantly renal expression of AE4 (377). In microdissected preparations of rat kidneys, AE4 transcripts are most abundant in the cortical collecting duct (498). AE4 mRNA and immunoreactivity are also detected in the rat renal collecting duct cell line RCCD1 (798). Reports, individually considered in Appendix VIII, conflict as to the precise location of AE4 protein within the collecting

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The Nt domain of AE4 includes a leucine-zipper– consensus sequence that may contribute towards protein oligomerization or interactions with binding partners (498, 720). The third extracellular loop includes the four cysteines that are common to NCBTs, and includes multiple putative glycosylation sites, although an in vitro study failed to demonstrate N-glycosylation of human AE4 (720).

MARK D. PARKER AND WALTER F. BORON duct. Although the consensus is that the AE4 protein is expressed predominantly in intercalated cells, it is not agreed whether AE4 is located in ␣- vs ␤-intercalated cells (see Ref. 498 versus Refs. 87, 982, and 1054). There is also controversy as to whether AE4 is localized to the apical versus basolateral membrane (see Refs. 762, 982, and 1054 versus Refs. 87 and 498), although we note that the data pointing to an apical location are all from rabbits. These disparate observations have been suggested to represent interspecific differences, although they might equally be explained by two other factors.

2) Putative sites of AE4 expression are designated as either ␣- or ␤-intercalated cell subtypes, but none of the studies considers the substantial subpopulation of intercalated cells in the collecting ducts of mice, rabbits, and rats that are non-␣/non-␤ types (270, 487).84 The evidence discussed in Appendix VIII is consistent with the hypothesis that AE4 is expressed in the basolateral membranes of both ␤-intercalated cells and non-␣/non-␤ intercalated cells (collectively known as non-␣ types) in rats and mice. The expression of AE4 in ␣-intercalated cells is not well demonstrated. It is unclear why anti-AE4 antibodies immunoreact with epitopes in the apical membranes of rabbit intercalated cells. L) REPRODUCTIVE SYSTEM.

An NCBI-curated EST database suggests that human and mouse testes are a minor site of AE4 transcription (Appendix VI). 7. Physiological roles Inasmuch as the expression of AE4 is mainly restricted to the kidney and inasmuch as AE4-null mice do not have an obvious renal phenotype, the physiological role(s) of AE4 is presently unknown. Four possible roles for AE4 have been proposed.

I) Suggested role in gastric HCO3⫺ secretion. A 2003 study reported AE4 immunoreactivity in the apical membranes of mouse stomach epithelia, where A) UPPER DIGESTIVE SYSTEM.

84 ⫺ (i.e., lumen to blood) ␣-Type intercalated cells reabsorb HCO3 and are characterized immunologically by an apical presence of a vacuolar-type H⫹ pump and a basolateral presence of AE1 (mediat⫺ ⫺ efflux). ␤-Type intercalated cells secrete HCO3 (i.e., blood ing HCO3 to lumen) and are characterized immunologically by an apical pres⫺ ence of pendrin (Slc26a4, mediating HCO3 efflux), a basolateral presence of a vacuolar H⫹ pump, and a lack of basolateral AE1. Non-␣/non-␤-intercalated cells (sometimes refered to as ␥-subtypes) have an apical presence of pendrin and H⫹ pump and lack basolateral AE1.

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B) LOWER DIGESTIVE SYSTEM.

I) Suggested role in duodenal HCO3⫺ secretion. A 2003 study detected AE4 immunoreactivity in the apical membranes of mouse and rabbit duodenal epithelia where HCO3⫺ secretion mediated by an apical Cl-HCO3 exchanger might play a role in mucosal protection (87). However, 1) others do not detect AE4 transcripts in duodenal epithelial cells of mice (887), 2) Cl-HCO3 exchange is unperturbed in the apical membranes of duodenal villus cells from AE4-null mice (887), and 3) later work suggests that an Slc26a6 product is the apical Cl-HCO3 exchanger in these cells (887). Thus, if AE4 is indeed in the apical membranes of these cells, its role remains undemonstrated. I) Suggested role in support of renal H⫹ secretion. The apparent localization of AE4 to the basolateral membranes of collecting-duct ␣-intercalated cells led to the suggestion that AE4 (acting as a Cl-HCO3 exchanger) could act in parallel with basolateral AE1 to support H⫹ secretion (498). However, experimental evidence for such a physiological role is lacking, inasmuch as the presence of AE4 in ␣-intercalated cells is not well demonstrated, and AE4 protein levels are not compensatorily increased in AE1 null-mice (904).85 C) URINARY SYSTEM.

II) Suggested role in support of renal HCO3⫺ reabsorption in acidosis. A preliminary study suggests that, in collecting duct ␤-intercalated cells isolated from rabbits (see footnote 84), acidosis reduces the abundance not only of pendrin at the apical membrane but also the abundance of AE4 mRNA as well as the abundance of AE4 immunoreactivity in a subapical compartment (762). Inasmuch as ␤ cells mediate transepithelial HCO3⫺ secretion and pendrin mediates the apical step (HCO3⫺ efflux into lumen), it is tempting to speculate that AE4 might normally contribute to HCO3⫺ secretion in rabbits. If this speculation is correct, and if the AE4 protein is present in the apical membrane of these cells, then AE4 would have to be a Cl-HCO3 exchanger. On the other hand, if AE4 exhibits a basolateral distribution in rabbit ␤-intercalated cells, as is the case in other model animals, then AE4 would have to mediate HCO3⫺ transport coupled to Na⫹ influx (i.e., Na/HCO3 cotransport), rather than to Cl⫺ efflux (i.e., Cl-HCO3 exchange), to contribute

85 The defect in AE1-null mice may be partly compensated by upregulation of the AE1-colocalized transporter Slc26a7, which some investigators describe as a Cl-HCO3 exchanger (921) (see footnote 2).

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1) None of the anti-AE4 antibodies that are considered in Appendix VIII recognizes a single band of the appropriate molecular weight in western blots of kidney preparations. Thus the specificity of these antibodies is not demonstrated.

HCO3⫺ secretion mediated by an apical DIDS-sensitive ClHCO3 exchanger would contribute to mucosal protection (87). However, later work by the same group suggests that an Slc26a9 product, not an Slc4a9 product, is responsible for the apical Cl-HCO3 exchange activity in stomach epithelia (1055). Thus, if AE4 is indeed at the apical membrane of these cells, its role is unclear.

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

to HCO3⫺ secretion. We note that NCBT activity has not been reported in ␤-intercalated cells. 8. Causes of AE4 upregulation A) CIRCULATORY SYSTEM.

I) Increased protein abundance in ischemia/reperfusion. Haqqani et al. (366) used a proteomic approach (ICAT-nanoLC-MS/MS) to identify proteins, among them AE4, whose expression level was altered in the microvasculature of rats following global cerebral ischemia and reperfusion (366). Microvascular AE4 protein abundance was transiently increased 1 h after ischemia/ reperfusion but returned close to normal levels after 6 h. The physiological relevance of this phenomenon is unknown.

A) URINARY SYSTEM. I) Downregulation in Foxi1-deficient mice. Foxi1-deficient mice lack properly differentiated ␣and ␤-intercalated cells, are afflicted with distal renal tubular acidosis, and not surprisingly lack renal AE4 (87). Due to the morphological abnormalities in the tubules, and because Foxi1-null mice also lack AE1 and the H⫹-pump subunit ATP6B1, defects in either of which alone is sufficient to cause dRTA (130, 473), the acidosis in these mice cannot be uniquely linked to the loss of AE4.

II) Decreased mRNA abundance and disturbance of AE4 protein distribution in acidosis. A preliminary study suggests that in collecting duct ␤-intercalated cells acidosis reduces the abundance not only of pendrin at the apical membrane but also the abundance of AE4 mRNA as well as the basolateral presence of AE4 protein (762). 10. Consequences of AE4 dysfunction There are no reports of pathologies linked to the SLC4A9 locus in humans, nor are we aware of any reports of phenotypical consequences related to the loss of AE4 in mice.

C. BTR1 (Slc4a11)

2. Nomenclature BTR1, bicarbonate transporter related protein 1 (720), is the most divergent member of the vertebrate Slc4 family and also the last member to be cloned. An alternative name, NaBC1, Na-coupled borate cotransporter 1 (712), was proposed following a report that BTR1 is a borate transporter. For the purposes of the present review, we will continue to refer to Slc4a11 products as BTR1 because NaBC1 is unfortunately similar in name to NABC1, a breast cancerassociated gene (198) that is located on the same human chromosome as SLC4A11, and the acronym NaBC1 does not usefully distinguish Na-borate cotransporters from Nabicarbonate cotransporters. 3. Molecular action The function of mammalian BTR1 as a borate transporter remains controversial. One group suggests that BTR1 has a dual action (712). In the absence of borate, BTR1 is proposed to function as an electrogenic Na/OH cotransporter, or Na-H exchanger, which is thermodynamically equivalent, that carries two or more OH⫺ per Na⫹. However, in the presence of borate, BTR1 is proposed to function as an electrogenic Na/B(OH)4 cotransporter that carries two or more Na⫹ per borate, a Na/anion stoichiometry that is opposite to that for Na/OH cotransport. Three main observations support the borate-independent action of BTR1.

1. Summary In mammalian genomes, Slc4a11 is the singleton representative of a third subgroup of Slc4 genes. The exon boundaries of Slc4a11 are distinct from those of genes that encode NCBTs and AEs (FIGURE 7). The demonstration that Slc4like family members from Arabidopsis thaliana (BOR1) and Saccharomyces cerevisiae (Bor1p) promote boron efflux from cells (943) led to the suggestion that BTR1, too, is a boron transporter (299). However, the apparent clustering of BOR1 and Bor1p with mammalian BTR1 protein sequences on a cladogram (299) owes more to their collective lack of identity to mammalian AEs and NBCs than any

1) BTR1-expressing HEK-293 cells acidify to a greater extent than control cells upon removal of extracellular Na⫹. This NHE-like activity in BTR1-expressing cells is not blocked by 10 ␮M EIPA nor by 500 ␮M DIDS. These cells also acidify in response to removal of extracellular K⫹, which could either be interpreted as K-H exchange or, because that maneuver would tend to hyperpolarize these cells, outward electrogenic Na/OH cotransport. 2) BTR1 expression reduces the ability of HEK-293 cells to defend pHi from increases or decreases in pHo, as if the cells have an increased flux of OH–/H⫹.

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9. Causes of AE4 downregulation

specific identity among BOR1, Bor1p, and BTR1 (TABLE 3). Nonetheless, a subsequent report provided indirect evidence that BTR1 is an electrogenic Na/borate cotransporter (712). Although BTR1 exhibits a wide distribution, genetic defects in BTR1 so far are associated only with a number of corneal dystrophies. Despite the uniqueness of BTR1, there is much to be learned from BTR1 about AEs and NCBTs. For example, pathological mutations occur at many of the same conserved sequence positions in BTR1, AE1, and NBCe1. Thus the numerous mutations that have been described in BTR1, but not (yet) in NBCe1, could point at critical residues for NBCe1 structure-function analysis.

MARK D. PARKER AND WALTER F. BORON 3) Elevating [K⫹]o causes a reduction of [Na⫹]i in BTR1expressing cells. Assuming that the rise in [K⫹]o shifts Vm in the positive direction, we do not understand how this observation can be interpreted in light of the electrogenic Na/OH model (point 1), which should, by itself, have caused [Na⫹]i to rise. Three main observations support the borate-dependent action of BTR1.

2) The presence of borate stimulates a small inward current in BTR1-expressing HEK-293 cells and oocytes. 3) In the presence of borate, the removal of extracellular Na⫹ elicits a larger outward current in BTR1-expressing HEK-293 cells and oocytes than in control cells. It is this result that requires a Na⫹:borate stoichiometry greater than 1:1. It must be noted that the above study presents no direct evidence of borate flux. Moreover, the concentrations of borate (i.e., 5 mM) are two orders of magnitude greater than physiological concentrations of this trace element (plasma [B] values are reviewed in Ref. 403). Finally, we are surprised that that BTR1 would bind 1 Na⫹ plus 2 anions in the absence of borate but 2 Na⫹ plus 1 anion in the presence of borate. Another group (1005), in a paper about BTR1 trafficking, reports (citing unpublished data) that they are unable to replicate the data of the previous group. At present, no data are available concerning the ability of AEs or NCBTs to transport borate in place of bicarbonate or carbonate. Likewise, the ability of BTR1 to transport HCO3⫺ has not been directly measured. 4. Genome The human SLC4A11 gene, which contains 20 exons that occupy 12 kb on chromosome 20 (720), is the shortest of the 10 mammalian Slc4 genes. The gene locus was originally assigned to position 20p12 (720), but subsequent refinements of the human genome map now place SLC4A11 at 20p13. The absence of a TATA-box and presence of a downstream promoter-element–like sequence suggests SLC4A11 gene expression is under the control of a TATA-less promoter (720).

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Three full-length transcripts are reported to be transcribed from the SLC4A11 gene. The three differ in the inclusion of alternative extreme Nt sequences. The archetypal BTR1 sequence, which we provisionally term BTR1-a, is a 3.1-kb transcript derived from exons 3–20 of SLC4A11. In BTR1-b, 30 amino acids encoded by part of exon 3 in BTR1-a are replaced by 57 amino acids encoded by exon 2. In BTR1-c, the 30 amino acids of BTR1-a are replaced by 14 amino acids encoded by exon 1. In theory, any of the aforementioned transcripts could encode two proteins variants of ⬃100 kDa, inasmuch as there are two initiating methionine codons, corresponding to Met1 and Met36 of BTR1-a. The second of the two start codons is preceded by the consensus Kozak sequence “CCACC.” Interestingly, the shorter variant in each case would begin Met-Ser-Gln-Xaa-Gly; the same sequence that initiates from Met1 in BTR1-a. It is unknown what fraction of protein product expressed from BTR1-a mRNA initiates with Met1 versus Met36. Hydropathy analysis predicts that BTR1 has a similar topology to other Slc4 family members (720, 1005). The Nt domain is shorter than most other Slc4 members and contains a number of consensus PKA and PKC phosphorylation sites, together with an unusually high proportion, for an Slc4, of cysteine residues. As noted above, BTR1 does not appear to be blocked by DIDS (712). On the other hand, the BTR1 protein binds to H2DIDS and SITS affinity columns (1005), and BTR1 does include a putative DIDS-interaction motif “KGTVK” at the extracellular end of TM5. Cell-free translation in canine pancreatic microsomes (720) and western blots of BTR1 expressed in HEK-293 cells (1005, 1010) reveal that BTR1 is N-glycosylated on at least one of the two consensus glycosylation sites in its third extracellular loop. This loop lacks the four conserved cysteines characteristic of NCBTs. 6. Distribution BTR1 expression is widespread and in many cases is expressed in cell types that also express NCBTs. The distribution of BTR1 in specific organ systems is discussed below. A) CENTRAL NERVOUS SYSTEM. I) Brain. BTR1 transcripts have been detected in mouse whole brain preparations (378) but not in rat cerebellum preparations (755). It is possible that the brain BTR1 transcripts are derived from choroid plexus, rather than neurons or glia (see next section).

II) Choroid plexus. BTR1 immunoreactivity is evident in the apical membranes of human choroid plexus epithelial

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1) In BTR1-expressing HEK-293 cells, the presence of “borate, i.e., an equilibrated mixture of H2BO3 and the anion B(OH)4–, exaggerates the pHi decrease caused by removing extracellular Na⫹ or by lowering pHo. If we assume that H3BO3 is freely diffusible, this acidification would be expected if B(OH)4– export caused the intracellular reaction H3BO3 ⫹ H2O ^ B(OH)4– ⫹ H⫹ (pK ⬵9.2) to shift to the right.

5. Structural features and variants

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

cells, although BTR1 transcripts are reportedly absent from mouse choroid plexus (755).

G) LOWER DIGESTIVE SYSTEM.

B) SENSORY ORGANS. I) Eye. BTR1 transcripts (1011) and protein (214, 608) are detected in corneal endothelium, as is a BTR1-lacZ fusion protein86 in a transgenic mouse (340). The polarity of BTR1 expression in these cells has not been described, and thus BTR1 is not shown in FIGURE 19. The absence of BTR1 transcripts has been reported in the human retina (see supplemental information in Ref. 1011). Except for a weak and occasional presence in the anterior corneal squamous epithelium (214, 608),87 the presence of BTR1 has not been reported in any other ocular structures.

II) Liver. According to an NCBI-curated EST database, the mouse liver is a site of BTR1 transcription (Appendix VI). III) Intestines. BTR1 transcripts have been detected in preparations of ileum and jejunum from pigs (583) and from colonic extracts from mice (512). H) ENDOCRINE SYSTEM.

We are aware of no reports of BTR1 expression in the endocrine system. I) LYMPHATIC AND IMMUNE SYSTEMS. I) Bone marrow. According to an NCBI-curated EST database, the bone marrow of mice is a site of BTR1 transcription (Appendix VI).

II) Spleen. BTR1 protein has been detected in extracts from rat spleen (712). J) URINARY SYSTEM.

BTR1 transcripts (720, 755, 1011) and protein (712) are abundant in whole kidney extracts and in the HEK-293 and MDCK renal cell lines (712).

C) RESPIRATORY SYSTEM.

BTR1 transcripts have been detected in RNA preparations from human trachea (720). According to an NCBI-curated database of ESTs, the olfactory mucosa of mice and lung of humans and mice are additional sites of BTR1 transcription (Appendix VI).

In the renal cortex, BTR1 immunoreactivity is demonstrated in glomerular podocytes and in the basolateral membranes of proximal tubule epithelia (214). Immunoreactivity also is reported in the apical membranes of cortical collecting duct epithelia (214).

D) CIRCULATORY SYSTEM.

In the renal medulla, BTR1 transcripts are detected in the inner medulla, including preparations that are enriched in IMCDs (986) and in microdissected segments of the thin descending (754) and thick ascending (694) limbs of the loop of Henle. In outer medullary collecting ducts, BTR1 immunoreactivity is reported in the apical membranes of intercalated cells. However, in inner medullarly collecting ducts, BTR1 is in the basolateral membranes of intercalated cells (214).88

According to an NCBI-curated EST database, BTR1 transcripts are present in RNA preparations derived from human blood (Appendix VI). BTR1 immunoreactivity is present in blood vessels in rat submandibular salivary glands (712). E) MUSCULOSKELETAL SYSTEM. We are unaware of reports of BTR1 expression in the musculoskeletal system. F) UPPER DIGESTIVE SYSTEM. BTR1 transcripts have been detected in salivary gland extracts (720). BTR1 protein has been detected in both parotid and submandibular salivary gland preparations from rats and mice as well as in a rat submandibular cell line (712). In rat submandibular glands, BTR1 immunoreactivity is reported to be in the basolateral membranes of acinar cells (i.e., in the same membrane as NBCe1 in FIGURE 21A) but not duct cells (712).

86 A soluble, ⬃320-amino acid fragment of the BTR1 NH2 terminus fused to ␤-galactosidase. 87 Groger and co-workers find no evidence of BTR1 expression in the anterior corneal epithelium in mice using an anti-BTR1 antibody (340), nor is BTR1 promoter activity disclosed in these cells by ␤-galactosidase assays of corneal sections from BTR1-lacZ transgenic mice (340).

A different renal distribution of BTR1 gene expression is suggested by studies of transgenic mice that express a BTR1-lacZ fusion protein. In these mice, the ␤-galactosidase reporter activity is absent from renal cortex, but present in the renal papilla and also in structures that are reported, by process of elimination, to represent the thin descending limbs of Henle’s loop (340). Thus the antibody and the lacZ data are consistently positive for BTR1 only in the case of the thin descending limb.

88 BTR1 transcripts have been detected in inner medullary preparations, apparently decreasing in abundance in segments closest to the papilla, according to a semiquantitative study (754), but oddly were undetectable in microdissected inner medullary duct preparations (754).

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II) Ear. BTR1 immunoreactivity is detected throughout the inner ear, specifically in the fibrocytes of the spiral ligament that underlie the stria vascularis of the cochlea (340, 608), together with a lesser presence in the spiral limbus of the cochlea and in the stroma that underlies the sensory epithelia of the macula of saccule in the vestibular system (608). According to an NCBI-curated database, more ESTs have been isolated from mouse inner ear preparations than from any other organ (Appendix VI).

I) Pancreas. BTR1 protein is detected in extracts from rat pancreas (712).

MARK D. PARKER AND WALTER F. BORON K) REPRODUCTIVE SYSTEM. BTR1 protein has been detected in a human cervical cancer cell line (712), and ESTs have been detected in preparation of human ovary (Appendix VI).

7. Physiological roles

The United States Department of Agriculture does not currently classify boron as an essential nutrient,89 but several nutritional studies suggest that boron deficiency can have detrimental consequences for mammalian physiology. The underlying cause to most of the pathologies associated with boron deficiency (reviewed in Refs. 402 and 403) is the increased activity of enzymes that are normally inhibited in the presence of boric acid. Such enzymes include serine proteases (e.g., those released by activated leukocytes) and vitamin D-24-hydroxylase (the enzyme that catalyzes the first step in the inactivation of vitamin D3). Thus “boron” has anti-inflammatory action and also potentiates the effects of vitamin D3, promoting Ca2⫹ reabsorption and increasing insulin sensitivity (402, 403). Furthermore, boric acid is a ligand for molecules such as ribose, S-adenosylmethionine, ATP, ADP, cAMP, NAD⫹, and NADH, although the consequences of boric acid binding for the bioactivity of these molecules has not been investigated (402, 403). Even given the usefulness of boron, it is reasonable to ask if a borate transporter would be useful in mammals. Dietary insufficiency of boron is rare. In fact, boron is so pervasive, and its normal dietary level so low, that it is technically challenging to reduce the boron content in animal feed to lower-than-normal levels. Although, boric acid is freely diffusible across artificial lipid bilayers (256), this observation does not address boric acid permeability of living cell membranes. In plants, aquaporins are responsible for boron uptake, whereas Slc4-like molecules are responsible for boron extrusion (FIGURE 9). Even if certain mammalian AQPs could provide a pathway for passive boric acid fluxes across membranes, BTR1 might still be useful for concentrating, or, alternatively, preventing toxic buildup of, boron inside cells.

89 Boron is one of 11 “ultratrace elements” that have a dietary requirement of ⬍1 ␮g/g body wt, and for which pathological consequences of dietary insufficiency have not been adequately demonstrated (683). Boron is, however, an essential nutrient for plants.

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8. Causes of BTR1 upregulation A) LOWER DIGESTIVE SYSTEM. I) Increased transcript abundance following dietary boron supplementation. In a study of pigs, a doubling of dietary boron content, maintained over 18 days, resulted in a threefold increase in BTR1 transcript abundance in jejunal preparations but no increase in ileal preparations (583). A quadrupling of normal dietary boron intake had no greater effect on BTR1 transcript abundance in either the ileum or jejunum (583). The consequence of the upregulation is not known, but is consistent with a role of BTR1 in boron homeostasis. If BTR1 was normally involved in boron secretion, upregulation of BTR1 would enhance boron loss under conditions of excess boron intake.

9. Causes of BTR1 downregulation A) SENSORY ORGANS. I) Reduced transcript abundance in the cochlea in response to acoustic trauma. A preliminary qPCR study reveals that BTR1 transcript abundance is reduced in the cochlear lateral wall of mice in response to acoustic trauma, an observation that the authors of that study link to a consequence of hypoxia (1073). B) LOWER DIGESTIVE SYSTEM. II) Reduced transcript abundance

following probiotic treatment. Probiotic treatment of mice is a model for investigating the molecular mechanism underlying the health benefits associated with probiotic treatment of inflammatory bowel disorder and ulcerative colitis. Kotka and co-workers (512) report a fourfold decrease in BTR1 transcript abundance in mouse colon 24 h after treatment with a probiotic mix (512). C) URINARY SYSTEM.

I) Decreased transcript abundance following dietary boron supplementation. In the same study of pigs as was mentioned above, a doubling of dietary boron content, maintained over 18 days, resulted in a twofold decrease (rather than the increase observed in the jejunum) in renal BTR1 transcript abundance (583). A quadrupling of normal dietary boron intake had no greater effect on renal BTR1 transcript abundance (583). The consequence of the downregulation is not known, but is consistent with a role of renal BTR1 in boron homeostasis. If BTR1 was normally involved in boron reabsorption, downregulation of BTR1 would enhance urinary boron loss under conditions of excess boron intake.

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BTR1 has no demonstrated physiological role, although as discussed in the following sections, defective BTR1 expression is associated with a number of pathologies. Assigning a hypothetical role for BTR1 is difficult because 1) no consensus has yet been reached concerning the molecular action or polarized distribution of BTR1 and 2) it is unlikely that the human pathologies associated with BTR1 defects are solely the result of a functional deficit in BTR1. However, we can at least speculate on the role of borate transport, the suggested role of BTR1.

Studies that address the ability of mammalian organs to accumulate, or defend themselves from overaccumulation, of boron are difficult to reconcile among themselves because of differences among species, and individuals of different maturity. However, these studies provide indications that boron is not passively distributed throughout the body but instead is selectively accumulated or eliminated by certain cell types (781). There is insufficient data to determine whether BTR1 is involved in these processes.

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

10. Consequences of BTR1 dysfunction A) GENERAL. I) Cell proliferation. One study found that siRNA knockdown of BTR1 in HeLa cells resulted in a reduction in cell proliferation that could be rescued by increasing the concentration of extracellular boron (712). In individuals with SLC4A11 defects, a decreased proliferative ability may be expected to contribute towards the severity of dystrophies involving cell types in which BTR1 is normally expressed. B) SENSORY ORGANS.

BTR1 is abundantly expressed in the corneal endothelium, a monolayer of squamous/low-cuboidal epithelial cells that forms the inner surface of the cornea. The endothelium plays a role in maintaining stromal deturgescence (i.e., corneal transparency) by reabsorbing fluid that moves by osmosis from the aqueous humor into the stroma. This reabsorption prevents disruption of the transparent crystalline array of collagen fibers and proteoglycans that constitute the stromal matrix (267). The manifestations of the dystrophy are a loss of endothelial cell density, stromal thickening, corneal clouding, and visual impairment (reviewed in Ref. 496). Interestingly, gene-trap disruption of Slc4a11 in mice (caused by the random insertion of a neomycin-resistance cDNA) causes no major corneal phenotype, other than a slight thickening of the basal cell layer of the corneal anterior epithelium (608). A separate study of transgenic mice in which Slc4a11 was disrupted with ␤-galactosidase also revealed a slight thickening of the epithelium as well as a doubled thickness of the corneal endothelium, Descemet’s membrane, and stroma (340). Furthermore, the endothelial layer was vacuolized and the stroma included Na- and Clenriched crystalline deposits (340). The clarity (or lack 90 A database of SLC4A11 mutations is curated at the Leiden Open Variation Database (https://grenada.lumc.nl/LOVD2/mendelian_ genes/home.php?select_db⫽SLC4A11).

An alternate possibility is that the absence of transport function (whatever the nature of that function) is not the main cause of the corneal endothelial dystrophy in humans with mutations in SLC4A11. With a few possible exceptions identified in individuals with late-onset dystrophy (794), all of the human mutant BTR1 proteins tested to date accumulate in the ER when overexpressed in mammalian cells (1005, 1011, 1012). It is possible, as has been demonstrated for other mutant proteins that misfold in the corneal endothelium (274), that the expression of large amounts of misfolded mutant protein causes ER stress (the “misfolded protein response”) and ultimately death of corneal endothelial cells. However, this hypothesis remains to be tested. II) Hearing: loss. Slc4a11-null mice exhibit a reduced response to auditory stimuli, an observation that is consistent with the genetic link between mutations in SLC4A11 and the perceptive deafness associated with Harboyan syndrome (340, 608). C) URINARY SYSTEM. I) Polyuria. A study of mice in which Slc4a11 was disrupted with a lacZ gene revealed that these mutant mice excrete more urine per day than wild-type mice and that the urine of mutant mice has a lower osmolarity and [Ca2⫹] compared with that excreted by wild-type mice (340). Gröger and co-workers explain the polyuria by suggesting that BTR1, in the thin descending limb of the loop of Henle, normally mediates Na⫹ influx and thereby contributes towards the efficacy of the countercurrent multiplier. However, no direct evidence for such a role is provided in that study, and the role of BTR1 elsewhere in the kidney is not considered. Furthermore, the authors report a significant decrease in NKCC2 mRNA in one of two data sets from these mice (see Supplementary Table 2 that accompanies Ref. 340), an alteration that could potentially contribute to a polyuric phenotype (939).

VII. CONCLUDING REMARKS A. Summary In this section we consider the common themes that emerge as we revisit the structure, actions, and roles of the five NCBTs. Our consideration reveals a number of unresolved issues as well as several emerging topics that are understudied. In this section we summarize these points using the

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I) Vision: corneal dystrophy. To date, nearly 60 mutations90 have been identified, scattered across the length of the BTR1 molecule, among individuals with corneal dystrophies (19, 20, 161, 244, 375, 450, 522, 640, 782, 794, 867, 919, 1011, 1012). Pathological SLC4A11 mutations are most frequently inherited in a homozygous recessive manner, although numerous cases of compound heterozygous inheritance of SLC4A11 mutations have been described (20, 244, 375, 782, 919). There are three SLC4A11-associated corneal endothelial dystrophies: 1) congenital hereditary endothelial dystrophy (CHED2), first associated with SLC4A11 in Reference 1011; 2) Harboyan syndrome, also known as corneal dystrophy and perceptive deafness (CDPD), first associated with SLC4A11 in Reference 244; and 3) late-onset Fuchs’ endothelial corneal dystrophy (FECD4), first associated with SLC4A11 in References 330 and 1012.

thereof) of the mouse cornea was not reported in this study. Thus neither of these mice are demonstrated to adequately model all of the features of human corneal dystrophy. Others have suggested that these gene-disrupted mice do not have a clear CHED phenotype due to factors such as the proliferative ability of mouse endothelial cells, functional redundancy with other transporters, incomplete inactivation of the gene, or other undefined mouse/human differences (340, 608, 1012).

MARK D. PARKER AND WALTER F. BORON same subject areas that we used in organizing sections V and VI.

B. Nomenclature 1. Nonmammalian

2. Mammalian In the mammalian realm, the initially confusing nomenclature is now generally settled both for the transporters themselves (NBCe1, NBCe2, NBCn1, and NDCBE) as well as for the variants of each (e.g., NBCe1-A, NBCe1-B). However, the nomenclature for Slc4a9, Slc4a10, and Slc4a11 products is controversial because the molecular actions of these transporters, which determines their acronyms, is not universally agreed upon. For example, AE4 does not mediate Cl-HCO3 exchange in the hands of all investigators, nor does BTR1/NaBC1 mediate boron transport in the hands of all investigators. Furthermore, the action of the Slc4a10 product is reportedly different for the human (“NBCn2”) versus the mouse and rat (“NCBE”) protein. Until these matters are resolved, caution must be exercised in the use of these acronyms. We recommend that papers on mammalian clones always refer at least once, preferably in a prominent way near the beginning of the manuscript, to the Slc4 designation. Investigators in doubt about the phenotype might use the Slc4 designation exclusively.

C. Molecular Action The first two topics in this section deal with the diversity of NCBT gene products, either multiple NCBTs performing different actions, or multiple NCBTs performing the same action.

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Among the five mammalian NCBTs, are engendered at least four distinct molecular actions: 1) electrogenic Na/HCO3 cotransport, 2) electroneutral Na/HCO3 cotransport, 3) electroneutral Na/HCO3 cotransport with a HCO3⫺-independent conductance, and 4) Na⫹-driven Cl-HCO3 exchange. Each action has its own physiological niche. In the kidney, the basolateral step of HCO3⫺ reabsorption could not be effected by an electroneutral NCBT, which, driven by prevailing ion gradients, would contribute to HCO3⫺ secretion. Only a Cl-HCO3 exchanger (e.g., AE2 in the TAL, and AE1 in the ␣-intercalated cells) driven by an inwardly directed Cl⫺ gradient, or an electrogenic NBC (i.e., proximal tubule) driven by Vm in addition to prevailing ion gradients, could contribute to HCO3⫺ reabsorption. Another example of the advantage of being electrogenic versus electroneutral is illustrated in the case of NBCe1 in neurons. Neuronal acidification, which could dampen neuronal excitability during repetitive firing, is avoided due to an NBCe1-mediated DIA because depolarization promotes electrogenic HCO3⫺ import. On the other hand, being electroneutral is sometimes advantageous. For example, electroneutral NCBTs can counter the effects of a whole body acidosis on neuronal pHi, and thereby maintain neuronal excitability, without influencing or being influenced by Vm. Being coupled to Cl⫺ transport also has important consequences. A study of NCBT action in nematode neurons indicates that Cl⫺ efflux mediated by the action of an NDCBE plays an important role in the maturation of the CNS: by lowering [Cl⫺]i, it make ECl more negative than Vm, rendering GABAergic and glycinergic signaling inhibitory. On the other hand, NBCn1, which is not coupled to Cl⫺, regulates neuronal pHi without influencing [Cl⫺]i. The only molecular action of NCBTs that has no currently demonstrated role is the HCO3⫺-independent conductance mediated by NBCn1. In theory, this conductance could make Vm more positive in neurons, influencing the action of ion channels and electrogenic transporters. For example, a more positive Vm in neurons would tend to inactivate voltage-gated Na⫹ channels, rendering these cells less excitable. Benefits of having NCBTs with functional redundancy. There appears to be no discernible difference in the molecular actions of NBCe1 and NBCe2 or between the net transport activities of human NBCn1 and NBCn2. It is unclear, for example, why NBCe1 could not take the place of NBCe2 in hepatocytes, or why NBCe2 could not take the place of NBCe1 in proximal tubules. However, genetic redundancy has obvious potential benefits in providing

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Even within the vertebrate realm, where the homology among Slc4-like genes is sufficiently high to permit adherence to a consistent system of trivial nomenclature, some investigators have created their own nomenclature. An example is the frog ortholog of BTR1, which has been dubbed XNBC2 (1102). Outside the vertebrate realm, where direct orthologs of the 10 vertebrate Slc4 genes simply do not exist, no consistent system of trivial nomenclature is possible, a situation exacerbated by the lack of functional data that would normally inform the nomenclature. These nomenclature issues will likely cause confusion as the body of literature expands. We recommend that each study of an Slc4-like gene includes reference to GenBank or Ensembl DNA and protein accession numbers, as well as a list of any previous terminologies applied to the same gene/gene products in related organisms by others. Of course, wherever possible, the guidelines laid down by the nomenclature committees that oversee the genomes of those organisms should be followed.

1. Benefits of having multiple NCBTs with distinct molecular actions

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

backup in the case of haploinsufficiency. An example may be CNS neurons that contain both NBCn1 and NBCn2 (195). Moreover, the presence of both NBCe1 and NBCe2 (or NBCn1 and NBCn2) provides the opportunity for differential regulation during development or in response to stresses, or differential expression in different parts of the cell. 2. Transporters with controversial action

Another aspect to bear in mind is that some transporters exhibit different molecular actions in difference species. For example, some Slc4 proteins from fishes exhibit conductive features that are not shared with their mammalian counterparts (e.g., trout versus human AE1, Ref. 285).

How does the molecular action of an electrogenic NCBT compare with that of an electroneutral NCBT? So far, all that is known of the determinants of electrogenicity versus electroneutrality is that critical amino acid residues reside in EL4. 4. Unusual features of NBCn1 Three questions arise when we consider the action of NBCn1. What is the relationship between the Na/HCO3 cotransport activity and the HCO3⫺-independent conductance of NBCn1? Why is NBCn1 relatively insensitive to blockade by DIDS, despite retaining a seemingly intact DIDS-binding motif on TM5? Furthermore, why does DIDS block the NCBT activity attributed to NBCn1 in mesenteric arteries and trigeminal ganglion neurons–is it possible that differences in posttranslational processing or local environment can impact DIDS sensitivity?

3. Novel substrates 5. Undetermined inhibitor binding sites Slc4-like proteins from invertebrate species have been suggested to transport non-HCO3⫺ species such as silicate and valproic acid, although direct evidence is lacking. Cl-HCO3⫺ versus CO32⫺ versus NaCO3–: even for extensively characterized NCBTs, there remain many key questions concerning molecular action. Do NCBTs carry HCO3⫺ or CO32⫺? Preliminary studies indicate that, at least in the case of NBCe1 and NDCBE as expressed in oocytes, CO32⫺ is the dominant, if not the only, carbon-containing substrate. With the assumption that a transporter carries CO32⫺, does the CO32⫺ move in the form of the NaCO3⫺ ion pair, as may be the case for the Na⫹-driven Cl-HCO3 exchanger in the intact squid giant axon? What is the mechanism by which electrogenic NCBTs appear to be able to switch between a 1:2 and a 1:3 stoichiometry? What is the relationship between the molecular mechanisms of the exchangers/antiporters (e.g., AEs, NDCBE) and the presumed cotransporters/symporters (e.g., NBCe1, NBCn1)? Could all Slc4s be antiporters? For example, the electroneutral

Although the binding site for reversible DIDS inhibition is well described for NBCe1, the irreversible binding determinants are unknown as are the binding determinants for other drugs such as tenidap. 6. The K/HCO3 cotransporter The protein(s) responsible for this activity are undescribed. Does an NCBT working in an unusual mode contribute? Intriguing in this regard is the observation that the Na⫹ versus Li⫹ specificity of certain NCBTs appears to be celltype specific. An alternative explanation is that K/HCO3 cotransport could be mediated by another transporter family (e.g., SLC12, which includes the KCC K/Cl cotransporters). 7. Three-dimensional structure Critical to understanding the molecular action of any NCBT will be a high-resolution three-dimensional structure together with molecular dynamic simulations. However, no high-resolution structure is available for any Slc4 or Slc4like protein.

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The molecular actions of Slc4a9 (AE4) and Slc4a11 (BTR1/ NaBC1) remain controversial, as does the action of SLC4A10/Slc4a10 (NBCn2/NCBE). NCBTs may behave differently in diverse heterologous expression systems and when heterologously versus natively expressed. All systems include endogenous factors (e.g., ion channels) that can interfere with electrophysiological measurements. The benefits of performing transporter characterization in a heterologous system are many, but it is important, where possible, to reconcile the transport properties and inhibitor profile of the heterologously expressed transporter with the properties of the native protein.

cotransport of one Na⫹ and one HCO3⫺ would be thermodynamically equivalent to, and difficult to distinguish from, the cotransport of two Na⫹ and one CO32⫺, or the exchange of one Na⫹ and one CO32⫺ (or 1 NaCO3– ion pair) for one HCO3⫺.

MARK D. PARKER AND WALTER F. BORON NBCe1, NBCn1, and NBCn2 all have the capacity to encode a Ct that terminates with a PDZ domain. Note that the Ct of the three AEs is not known to be variable.

D. Genome 1. Diversity Humans, mice, and rats have 10 Slc4 genes. Other mammals likely have the same number, but we have yet to truly appreciate the diversity of Slc4-like genes in nonmammalian species. 2. Gene clusters

3. Promoter characterization An emerging and underexplored area in the study of mammalian Slc4 genes is the mapping and characterization of Slc4 promoter regions, the understanding of which will impact our knowledge of the consequence of the use of alternative promoters, NCBT dysregulation in disease, and the factors that are responsible for altering NCBT abundance in response to diverse physiological and non-physiological stimuli.

E. Structural Features and Variants

3. Influence of the Nt upon the TMD An emerging theme is that many NCBT splice cassettes include docking sites for protein partners (e.g., IRBIT) that can influence NCBT activity. Furthermore, the Nt of NBCe1 appears to be a binding partner for the TMD of NBCe1, and this interaction is necessary for Na/HCO3 cotransport activity. There are a number of important mechanisms that have yet to be elucidated in this regard. For example, how do the Nt, the ASD, the AID, and IRBIT exert their effects on Na/HCO3 cotransport by the TMD? One possibility is that the cytosolic domain acts as a scaffold for the TMD and that structural rearrangements in the Nt influence the ability of the Nt to scaffold the TMD in a transport-competent conformation, as has been proposed for the cytosolic domain of a ClC from red alga (283). 4. Isolated Nt variants

1. Role of UTRs At the level of Slc4 transcripts, we do not yet understand the consequences of alternative 5=- and 3=-UTR inclusion. These sequences likely include many determinants that impact the stability and efficiency of translation of the transcript, such as miRNA target sites. Because NCBT overabundance is linked to the poor outcomes in cancer, heart disease, and stroke, understanding how to manipulate NCBT transcript abundance would be valuable. 2. The diversity of NCBT protein variants At the level of protein sequence, most NCBTs exhibit similar patterns of variation. For example, all NCBTs, with the current exception of NBCe2, have variants that include an autoinhibitory Nt appendage.

Working with multiple cDNA libraries, investigators have amplified transcripts predicted to encode an isolated NCBT Nt domain (i.e., without a TMD). Such transcripts are produced by the Slc4a7, Slc4a8, and Slc4a10 genes. The abundance, stability, and relevance of protein expressed from these transcripts has yet to be described. Because the Nt does not appear in Slc4 evolution until the emergence of animals, the isolated Nt transcripts may represent the vestigial expression of the original “isolated Nt” open-reading frame that was appended to the “isolated TMD” transporter gene. The origin and original function of the ancestral isolated Nt is unknown. Note that the archetypal-Nt gene product need not have had the same open reading frame as the modern Nt.

F. Distribution All NCBTs, with the possible exception of NBCe2, have variants that are stimulated by interaction with the soluble protein IRBIT. NBCe1, NBCn1, and NBCn2 all exhibit variation in their Nt loop region. Note that this region is not known to be variable in the three AEs.

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1. Overview The distribution of NCBTs in the mammalian body is likely broader than is presently appreciated. In some instances, the location, but not the identity, of an NCBT is known as is the case with the DIDS-sensitive electroneutral NCBT

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SLC4A7 and SLC4A10 are both neighbored by T-box transcription factor genes (FIGURES 31 AND 39), indicating a long-standing relationship between the two gene families. Furthermore, growth factor genes are often located at similar chromosomal loci to Slc4 genes (e.g., TGFA and SLC4A5, see Ref. 591).

The commonality between these gene variations is evident when we compare the primary structure of the variants side by side as illustrated in Appendix V. Despite the wealth of variants encoded by the NCBTs genes, we can be certain, based on variation between EST sequences, that more are yet to be described. In most cases, the physiological consequence of such variance is unknown, as are the mechanisms that dictate the presence or absence of the splice cassettes in specific tissues.

⫺ TRANSPORTERS Na⫹-COUPLED HCO3

activity in platelets (315). A thorough analysis of NCBT distribution would require the use of variant-specific and variant-independent primers and antibodies that are still in development, together with their application in normal and stressed tissues that have yet to be probed. In this respect, the study of transgenic animals that express reporter genes under the control of NCBT promoters could be useful. 2. Apparently overlapping distribution

3. The polarity of NCBT expression With few exceptions, the epithelial polarity of NCBTs, and indeed all Slc4s, is basolateral, complementing the usually apical distribution of Slc26 proteins (FIGURE 1). Some unusual epithelia express basolateral markers in their apical membranes, two examples that pertain to NCBT expression being the choroid plexus (with apical NBCe2) and the retinal pigment epithelium (with apical NBCe1). However, other reports of apical NCBT expression ought to be regarded with caution, pending independent confirmation. One example is the unusual apical NBCn1 immunoreactivity in renal tubules disclosed by the “anti-NBC3” antibody, the use of which is documented in Appendix VII. This distribution has not been confirmed by the use of any other anti-NBCn1 antibodies, which react with basolateral targets. The reason for this disparity remains unclear.

G. Physiological Roles 1. Overview NCBTs fulfill three main roles: pHi regulation, HCO3⫺ secretion, and Na/HCO3 reabsorption. In most cell types NCBTs mediate Na⫹ and HCO3⫺ influx. Indeed, when expressed in the basolateral membranes of polarized cells (e.g., NBCn1 in salivary acinar cells in FIGURE 21), NCBTs support ion and fluid secretion. In several studies, this contribution becomes apparent only following inhibition of intracellular CAs, consistent with the idea that it is the CAs (e.g., in conjunction with Na-H exchangers) that are dominant in generating HCO3⫺ for secretion under unstimulated conditions. In the CPE, apical NBCe2 could support HCO3⫺ secretion by operating with an apparent 1:3 stoichiometry to support HCO3⫺ secretion across the apical membrane into the CSF (FIGURE 28). NBCe1, by operating with an apparent 1:3 stoichiometry in the basolateral membranes of PT epithelia(FIGURE 23), is the only NCBT demonstrated to support HCO3⫺ reabsorption.

2. Inferences from phenotypes of transgenic mice Many of the physiological roles ascribed to NCBTs are inferred from the signs of mice and humans with disrupted NCBT genes. A potential complication is that, in some cases, the pathological signs may have more to do with the expression of misfolded protein (e.g., a partial transmembrane domain) rather than the absence of the physiological NCBT function per se. The observed signs may also depend on the nature of the NCBT disruption and the genetic background of the disrupted gene. In this regard, it will be informative to observe the phenotypes of multiple strains of mice in which the Slc4 has been disrupted in diverse ways (e.g., knockout versus gene-trap versus knocked-in mutation). The unintended consequences of dysregulation of other genes could also contribute to the observed phenotype, in some cases requiring that the investigators systematically examine the expression and activity of other transporters expected to contribute to the phenotype.

H. Causes of Upregulation As depicted in Appendix V, NBCe1-B/C, NBCn1, NDCBEA/B, and NBCn2 all include both autoinhibitory domains and modules in their Nt appendage that are predicted to render them sensitive to stimulation by IRBIT. However, although the physiological cues that activate IRBIT with respect to NCBTs are unknown, the abundance of NBCe1, NBCn1, and NDCBE transcripts are all increased during acidosis. In addition, NBCe1 and NBCn1 protein abundance is increased during hypercapnia. These observations are consistent with the role of these NCBTs in maintaining pH within a narrow physiological range. Lacking from our current knowledge are the full details of the molecular mechanism(s) by which the NCBTs are upregulated by acidosis/hypercapnia. In principle, upregulation could occur at any or all of four levels: transcript abundance, total protein abundance, plasma-membrane protein abundance, and per-molecule protein activity. At least in the case of NBCe1-B, a pH-responsive element has been identified in the promoter region, and in the case of NBCe1-A, some of the signaling components that enhance HCO3⫺ reabsorption in response to respiratory acidosis have been elucidated (e.g., Refs. 824, 890, 1104, and 1107). Characterization of promoter regions and the signaling cascades that enhance functional expression of NCBTs would be helpful towards the goal of understanding how the activity of multiple transporters are coordinated in a cell type. For example, an emerging theme is that IRBIT stimulates ion and fluid secretion by coordinated upregulation of multiple transporters.

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Existing studies suggest that all five NCBTs are present in the central nervous system, the choroid plexus, and the kidney. The reason for such apparent redundancy is unknown. It is also unknown if two NCBTs coexpressed in the same cell type, such as NBCe1 and NBCn1 in duodenal villar cells (FIGURE 22), are capable of heterodimerizing to create a transporter with novel properties.

In neurons and glia, HCO3⫺ transport mediated by electrogenic NCBTs (the direction of which appears to depend upon prevailing Vm and ion gradients) tends to maintain neuronal excitability.

MARK D. PARKER AND WALTER F. BORON

Rather than stimulating transport per se, the action of CAs in the vicinity of NCBTs could minimize pH changes close to the plasma membrane, thereby minimizing adverse effects on the activities of other membrane proteins. The metabolon controversy is reviewed in Reference 102.

I. Causes of Downregulation The abundance of NBCe1 and NBCn1 protein falls under alkalotic conditions, consistent with a reduced requirement for reabsorption and cellular influx of HCO3⫺. The abundance of NBCe1, NBCn1, NDCBE, and NBCn2 protein falls under hypoxic conditions, consistent with energy conservation and possibly also reflecting a response to respiratory alkalosis in these animals. The perturbation of NBCe2 abundance in response to hypoxia/alkalosis has not been reported. With the exception of the pH-responsive element located within the NBCe1-B/C promoter, the mechanisms that result in reduced NCBT abundance remain to be elucidated. An emerging and underexplored theme is that NCBT abundance can be reduced by miRNAs.

J. Consequences of Dysfunction 1. Transgenic mice Recurring phenotypes observed in NCBT-null mice are acidosis (NBCe1 and NBCe2), hypertension (NBCn1 and NBCe2), reduced neuronal excitability (observed for NBCe2, NDCBE, NBCn2, and inferred for NBCe1), impaired CSF secretion (NBCe2 and NBCn2), and ocular defects (NBCe1, NBCe2, and NBCn1). In the majority of cases, these phenotypes accord well with the known distribution and inferred physiological roles of each NCBT. Somewhat surprising, given the abundance of NBCe1 in the pancreas, NBCe2 in the liver, and NBCn1 in the mTAL, NBCe1-null mice lack an obvious pancreatic phenotype,

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NBCn1-null mice lack an obvious renal phenotype, and NBCe2-null mice lack an obvious hepatic phenotype. Presumably these mice have upregulated unknown compensatory pathways or have not been subjected to the appropriate challenges. In the case of NBCe1-null mice, another possibility is that the animals, which die shortly after weaning, have not lived long enough to exhibit a phenotype. In this respect, studies of conditional and inducible knockouts, which have not been reported for any NCBT, will be illuminating, as should be the application of antisense RNA technology in animals. One potentially confounding aspect of existing NCBT knockout mice is the interpretation of their phenotypes. For example, as mentioned above, especially in the case of genetrapped mice, some phenotypes may be consequences of the expression of partial, misfolded NCBT protein rather than consequences of the absence of the NCBT activity per se. Such mice may be better models for the pathological consequences of NCBT mutation in humans because some signs in affected individuals may be specifically due to misfolded protein response. Studies of targeted transgenic mice that carry orthologs of human mutations, the NBCe1-mutant W516X mouse that mimics a human pRTA is the only example to date, are the most appropriate in regard to modeling pathologies associated with human disease. In terms of investigating the results of the loss of NCBT transport activity, a transgenic mouse that expresses a nonfunctional, yet full-length NCBT would perhaps provide the clearest picture. Attempts to disrupt an NCBT gene close to its initiator Met include the potential hazard of permitting normal transcription of alternative gene products. 2. Linkage studies Numerous GWAS studies implicate variation at NCBT gene loci with susceptibility to various traits and disorders, such as autism, substance abuse, hypertension, and cancer. These associations are consistent with known roles for NCBTs in control of neuronal excitability, Na⫹ reabsorption, and in countering the apoptotic effects of acidosis. These studies will need to be followed up with deep sequencing, the results of which would provide a statistically significant link (or lack thereof) of the trait to a specific SNP within an NCBT gene. The next step would be to study, in a heterologous system or transgenic animal, the effect of the SNP on the functional expression or regulation of that NCBT. In conclusion, it seems likely, given the widespread abundance of NCBTs, and the effects of pH on almost all physiological processes, that NCBT action modifies a wide array of complex genetic traits. Our current knowledge concerning the diverse molecular actions, distribution, regulation, physiological roles, and pathophysiological roles of NCBTs provides only a glimpse of their potential importance.

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On the topic of activating binding partners, CAs are proposed to bind to the Ct and EL4 of NBCe1 and to the Ct of NBCn1, forming a “metabolon.” The concept is that CAs supply HCO3⫺ to the outer face of the transporter and remove HCO3⫺ from the inner face of the transporter, or vice versa, depending on the direction of movement, thereby speeding transport. This theory remains controversial for four major reasons: 1) NBCe1 stimulation by CAs is observed only in some studies; 2) the binding of CAs to the Ct of NCBTs is observed only in some studies; 3) if, as preliminary data suggest, NCBTs transport CO32⫺, rather than HCO3⫺, it is not clear that CAs would enhance NCBT action substantially; and 4) modeling data suggest that CAs would have only a minor effect on HCO3⫺ or CO32⫺ transport rates (Ref. 342 and Rossana Occhipinti, personal communications).

⫺ Na⫹-COUPLED HCO3 TRANSPORTERS

For Appendices I–VIII, the online version of this article contains supplemental material.

Appendix I: Annotated Protein Sequence Alignments of Human SLC4s (See Figures 3 and 15) Appendix II: GenBank or Ensembl Protein Accession Numbers for Nonmammalian Slc4-like Transporters (See Figures 4 and 8)

Parker), DK30344 (to W. F. Boron), DK81567 (to W. F. Boron), NS18400 (to W. F. Boron), HD032573 (to Gabriel G. Haddad/project 2 and W. F. Boron), and HL090969 (to Alanna C. Morrison).

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

REFERENCES

Appendix IV: GenBank Protein Accession Numbers for Mammalian Slc4 Variants (See Sects, V and VI) Appendix V: Annotated Protein Sequence Alignments of Human NCBT Variants (See Sect. V) Appendix VI: The Distribution of Expressed Sequence Tags for Humans and Mouse NCBTs, AE4, and BTR1 (See Sects. V and VI) Appendix VII: Locations of “Anti-NBC3” Immunoreactivity (See Sect. V) Appendix VIII: Locations of Renal Anti-AE4 Immunoreactivity (See Sect. VI) ACKNOWLEDGMENTS We thank Dennis Brown for his patient editorial oversight. We thank the two reviewers for their thorough reading of the manuscript and their helpful comments. We also thank members of the Boron lab past and present as well as all those investigators whose studies and personal communications have contributed to fabric of this review. Address for reprint requests and other correspondence: M. D. Parker, Dept. of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970 (e-mail: mark.d. [email protected]).

GRANTS This work was supported by National Institutes of Health Grants EY021646 (to Michael L. Jennings and M. D.

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