SLC4 base (HCO3 , CO3 2 ) transporters ... - Renal Physiology

SLC4 base (HCO − 2− 

3 , CO3 

) transporters: classification, 

function, structure, genetic diseases, and knockout models 

Alexander Pushkin and Ira Kurtz 

Am J Physiol Renal Physiol 290:F580-F599, 2006. ; 

doi: 10.1152/ajprenal.00252.2005 

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Invited Review 

� 2� 

SLC4 base (HCO3 ,CO3) transporters: classification, function, structure, 

genetic diseases, and knockout models 

Alexander Pushkin and Ira Kurtz 

Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, California 

� 2 

Pushkin, Alexander, and Ira Kurtz. SLC4 base (HCO3 ,CO3 

� 

) transporters: 

classification, function, structure, genetic diseases, and knockout models. Am J 

Physiol Renal Physiol 290: F580–F599, 2006; doi:10.1152/ajprenal.00252.2005.— 

In prokaryotic and eukaryotic organisms, biochemical and physiological processes 

are sensitive to changes in H� activity. For these processes to function optimally, 

a variety of proteins have evolved that transport H� /base equivalents across cell 

and organelle membranes, thereby maintaining the pH of various intracellular and 

extracellular compartments within specific limits. The SLC4 family of base 

� 2� 

(HCO3 ,CO3) 

transport proteins plays an essential role in mediating Na�- and/or 

Cl�-dependent base transport in various tissues and cell types in mammals. In 

addition to pH regulation, specific members of this family also contribute to 

vectorial transepithelial base transport in several organ systems including the 

kidney, pancreas, and eye. The importance of these transporters in mammalian cell 

biology is highlighted by the phenotypic abnormalities resulting from spontaneous 

SLC4 mutations in humans and targeted deletions in murine knockout models. This 

review focuses on recent advances in our understanding of the molecular organization 

and functional properties of SLC4 transporters and their role in disease. 

bicarbonate; transport 

� 2 

MEMBRANE PROTEINS THAT MEDIATE base (HCO3 ,CO3 

� 

) transport 

play an important role in eukaryotes in maintaining both 

intracellular pH (pHi) and extracellular pH (pHo) within nar- 

� 

row limits. This is not surprising given that the HCO3 / 

2� CO 3 

/CO2 buffer system is arguably the most important buffer 

in eukaryotic organisms. In mammals, two unrelated multigene 

families, SLC4 and SLC26 transporters, have evolved, whose 

members transport base in both a cell type- and cell membranespecific 

fashion. This review focuses specifically on the SLC4 

transporter family. The reader is referred to recent reviews on 

SLC26 transporters for an update on the role these transporters 

play in health and disease states (46, 131). Following an initial 

overview of the classification of the SLC4 family of transport 

proteins, we highlight recent advances in our understanding of 

the structure and function of these transporters and the role of 

protein interactions in the regulation of their functional properties. 

Abnormalities in either the function and/or membrane 

targeting of certain members of the SLC4 family are the cause 

of various genetic diseases in humans. Mouse knockout models 

have also provided a useful adjunct with which to study the 

role of SLC4 family members in specific organ systems. 

Advances in our understanding of the role of SLC4 transporters 

in health and disease are also the focus of this review. 

CLASSIFICATION OF SLC4 TRANSPORTERS 

Based on the nature of the specific transport process involved, 

e.g., exchange, cotransport, and the specific ions that 

are transported, SLC4 family members can be conveniently 

Address for reprint requests and other correspondence: I. Kurtz, Div. of 

Nephrology, David Geffen School of Medicine at UCLA, 10833 Le Conte 

Ave., Rm. 7–155 Factor Bldg., Los Angeles, CA 90095 (e-mail: 

ikurtz@mednet.ucla.edu). 

F580 

separated into three functional groups with potential transport 

� 2 

modes for HCO3 and/or CO3 

� 

(Fig. 1): 1) Na�-independent Cl� � 

/HCO3 exchangers mediating electroneutral exchange of 

Cl� � 2 

for base (HCO3 ,CO3 

� 

); 2) Na� � 

-HCO3 cotransporters 

mediating cotransport of Na� � 2 

and base (HCO3 ,CO3 

� 

); and 3) 

Na�-driven Cl� � 

/HCO3 exchangers mediating the electroneutral 

exchange of Cl� for Na� � 2 


� 

). SLC4 

proteins have in common the property of transporting base 

� 2 

(HCO3 , CO3 

� 

) 1 but differ in their ability to mediate the 

concomitant transport of Na � and or Cl � . In addition, the 

cotransport of Na � by most SLC4 transporters distinguishes 

this family from SLC26 proteins, which predominantly mediate 

Na � -independent anion transport. The sequences and classification 

of human SLC4 transporters are shown in Fig. 2 and 

Table 1. 

Na�-Independent Cl� � 

/HCO3 Exchangers 

SLC4A1 (AE1). AE1 mediates Na�-independent anion exchange 

in red blood cells and the renal collecting duct. Two 

variants of AE1 have been well characterized (6, 25, 33, 107, 

123, 196). Red cell AE1 (eAE1) or band 3 protein (human 

� 

eAE1, 911 aa) plays an important role in transporting HCO3 to 

the lung that was derived from metabolically produced CO2.In 

the kidney, AE1 (kAE1) is transcribed from an alternate 

is �500:1, whereas at a typical pHi of 7.1, 

is twice this value, or �1,000:1. It is currently unclear as to 

(or both) is transported in various SLC4 transporters. 

1 At pHo of 7.4, the HCO3 � /CO3 2 � 

Am J Physiol Renal Physiol 290: F580–F599, 2006; 

doi:10.1152/ajprenal.00252.2005. 

� 2 

the HCO3 /CO3 � 

� 2 

whether HCO3 or CO3 � 

The various potential transport modes are depicted in Fig. 1. At the present 

� 

time, the term “bicarbonate (HCO3 )” that is used in naming or describing the 

function (see Table 1) of SLC4 transporters should not be viewed as a 

definitive description of the type of base transported, but rather as a term that 

� 2 

generically refers to HCO3 and/or CO3 � 

until definitive studies are reported 

characterizing the specific species transported. 

0363-6127/06 $8.00 Copyright © 2006 the American Physiological Society http://www.ajprenal.org 




Fig. 1. SLC4 transporters can be divided functionally into 3 groups: Na�- independent Cl� � � � 

/HCO3 exchangers (A), Na -HCO3 cotransporters (B), and 

Na�-driven Cl� � � 

/HCO3 exchangers (C). Potential transport modes for HCO3 

2� 

and CO3 are depicted. In B,Na� � 

-HCO3 cotransporters are further subdivided: 

(i) electroneutral Na� � � 2� 

-HCO3 cotransporters with a HCO3 (CO3 )-to-Na� transport stoichiometry of 1:1, and electrogenic Na� � 

-HCO3 cotransporters 

� 2� 

with a HCO3 (CO3 

)-to-Na� transport stoichiometry of either 2:1 (ii) or3:1 

(iii) are shown. It has not been definitively established whether SLC4 proteins 

� 2� 

mediate HCO3 and/or CO3 transport. Depicted are the hypothetical modes of 

� 2� 

transport, wherein the coupling ratio of base (HCO3 and/or CO3 ) transport to 

Na � and/or Cl � is equivalent. Note that these transport modes are not 

equivalent with regard to the total flux of carbon, hydrogen, and oxygen per 

transport cycle. 

promoter, and the resultant protein has an NH2-terminal truncation 

(first 65 amino acids in human AE1) (25). Kidneyspecific 

AE1 is expressed on the basolateral membrane of 

collecting duct �-intercalated cells (7, 205), where it plays an 

important role in bicarbonate reabsorption and urinary 

acidification. 

SLC4A2 (AE2). AE2 mediates Na � -independent anion exchange 

and is more widely expressed than AE1. Five NH2terminal 

splice variants of AE2 (a, b1, b2, c1, and c2) have 

been described in mammals and are expressed in a tissue- and 

cell-specific manner (5, 6, 8–10, 13, 40, 110, 129, 168, 217). 

Human AE2 is �300 aa larger than eAE1 and, like AE1, is 

glycosylated (6, 233). Deglycosylation of AE2 does not appre- 

� 2� 

SLC4 BASE (HCO3 ,CO3) TRANSPORTERS 

AJP-Renal Physiol • VOL 290 • MARCH 2006 • www.ajprenal.org 


F581 

ciably affect its transport function (53). AE2 is widely expressed 

in nonexcitable tissues, where it has been proposed to 

be a housekeeping Cl � /HCO 3 � . It is also highly expressed on 

the basolateral membrane of choroid-plexus epithelial cells (9) 

and gastric parietal cells (191). In hepatocytes, AE2a, AE2b1, 

and AE2b2 have been localized to the apical membrane (13). 

In the kidney, AE2 is expressed in the basolateral membrane of 

the thick ascending limb (TAL) (6, 10). Ion transport mediated 

by AE2 expressed in Xenopus laevis oocytes (77, 188, 229) and 

mammalian cells (91, 115) is pH sensitive. Residues in the 

cytoplasmic NH2 terminus of mouse AE2, aa 336–347, 357– 

362, and 391–510, have been shown to regulate the pH dependence 

of the transporter (188–190, 229). 

SLC4A3 (AE3). AE3 mediates Na � -independent anion exchange 

like AE1 and AE2. Two NH2-terminal splice variants 

of AE3, cardiac (cAE3) and brain (bAE3), have been described 

(108, 118). The size of bAE3 (1,232 aa in humans) is comparable 

to AE2, whereas cAE3 is �200 aa shorter. Transcripts of 

both variants were found in different organs and tissues including 

the brain and heart (106, 108, 110, 118, 225). bAE3 protein 

has also been detected in basolateral membrane vesicles isolated 

from human ileal and colonic epithelial cells (11). AE3 is 

also expressed in the basal end foot processes of retinal Müller 

cells (106). AE3 expressed in X. laevis oocytes and HEK293 

cells mediates significantly less Cl � /HCO 3 � exchange than 

does AE1 or AE2 (53, 77, 115, 165, 225). Unlike AE2, when 

expressed in HEK cells, AE3 does not appear to be glycosylated 

(53). The decreased rate of AE3-mediated anion exchange 

in HEK cells has been attributed to a low level of 

plasma membrane targeting. cAE3 transport is stimulated at an 

alkaline pH similar to AE2 (188). 

Sodium Bicarbonate Cotransporters 

Sodium bicarbonate cotransporters mediate cotransport of 

Na� � 2 


� 

). 2 Depending on the flux of the 

net charge per transport cycle, these transporters are characterized 

� 

as electroneutral [i.e., transported negative charge (HCO3 and/or 

2 

CO3 � 

)] equals transported positive charge (Na� ), or electro- 

� 2 

genic [i.e., transported negative charge (HCO3 and/or CO3 

� 

)] 

exceeds transported positive charge (Na � ). 

SLC4A4 (NBC1, NBCe1). NBC1 is an electrogenic sodium 

bicarbonate cotransporter that was first cloned from Ambystoma 

tigrinum (166) and human (30) kidney. In humans, two 

major NBC1 variants, kNBC1 (NBCe1-A) (1,035 aa) and 

pNBC1 (NBCe1-B) (1,079 aa), that differ in their extreme NH2 

terminus are differentially expressed in a cell-specific manner 

(2) and are derived from alternate promoter usage in the 

SLC4A4 gene (4). A third NBC1 variant, rb2NBC (NBCe1c), 

has been cloned from rat brain and is identical to rat pNBC1 

except for 61 COOH-terminal residues and a COOH-terminal 

PDZ motif (18). Recently, a similar splice variant of the 

SLC4A4 gene has been detected in human and porcine vas 

2 As depicted in Fig. 1, in electrogenic sodium bicarbonate cotransporters, a 

� 

2:1 stoichiometry could result for example from the transport of 2HCO3 � 

1Na� 2 

, 1CO3 � 

� 1Na� � 2 

, or 2HCO3 � 1CO3 � 

� 2Na� . A 3:1 cotransporter 

� � stoichiometry could result from the transport of either 3HCO3 � 1Na , 

2 

3CO3 � 

� 2Na� � 2 

, or 1HCO3 � 1CO3 � 

� 1Na� . Although the total charge 

transported is identical, it is clear that the total carbon, hydrogen, and oxygen 

flux per transport cycle will differ and be dependent on the specific base 

species transported, e.g., HCO 3 � vs. CO3 2 � 

. 





F582 � 2� 


Fig. 2. Alignment of human SLC4 transporters: SLC4A1 (NP_000333), SLC4A2 (NP_003031), SLC4A3 (NP_005061), SLC4A4 (NP_003750), SLC4A5 

(NP_597812), SLC4A7 (NP_003606), SLC4A8 (NP_004849), SLC4A9 (NP_113655), and SLC4A10 (NP_071341). The alignment was performed using 

GeneWorks 2.5.1 software (Oxford Molecular Group, Campbell, CA). Putative transmembrane segments are shown in red (SLC4A1) and blue (SLC4A4) based 

on topological models (198, 232). Amino acids shown to be important for SLC4A1- and SLC4A4-mediated transport (1, 35, 87, 97, 132, 133, 195, 231) are shown 

in bold font. Amino acids important for plasma membrane expression of SLC4A1 (54, 231, 232) and SLC4A4 (1, 75, 116) are marked with brown stars. Lysine 

residues shown to covalently bind DIDS and H2DIDS in SLC4A1 (93, 140) or homologous lysine residues in SLC4A4 predicted to covalently bind 

DIDS/H2DIDS are marked with ❖. Note that AE2 (SLC4A2), which has been shown to be inhibitable by DIDS (6), has methionine (M 1181 ) in a second putative 

DIDS binding site instead of lysine, whereas NBC3 (SLC4A7), which has been shown to be insensitive to DIDS (155), has lysines in both putative DIDS binding 

sites. Glycosylation sites for SLC4A1 (33) and SLC4A4 (38) are depicted with a branched tree symbol. SLC4A1, SLC4A2, and SLC4A4 amino acids that have 

been shown to play critical roles in binding of carbonic anhydrase II (CAII) (68, 154, 163, 206–208) are shown in magenta. CAII binding sites are depicted with 

a magenta line. The COOH-terminal PKA phosphorylation site in NBC1 is shown in green (arrowhead). Note that the SLC4A11 transporter NaBC1 is not 

included in the alignment despite its distant homology to the other members of the SLC4 family, because recent studies have shown that it functions as a 

Na � -dependent borate transporter (142, 143, 145, 194). 





Table 1. Characteristics of SLC4 transporters 

deferens epithelial cells (148). NBC1 proteins have been 

shown to be glycosylated (38). kNBC1 is highly expressed on 

the basolateral membrane of the renal proximal tubule where it 

mediates sodium bicarbonate efflux (21, 127, 178, 223). In 

humans, the pNBC1 transcript is highly expressed in the 

pancreas and to a lesser extent in various other tissues (2). 

Unlike the kidney, wherein all studies agree on the localization 

of kNBC1, the expression pattern of pNBC1 in the 

pancreas is complex. NBC1 was localized with antibodies 

common to kNBC1 and pNBC1 to the basolateral membrane of 

large ducts in the human pancreas (125). In the rat pancreas, 

NBC1 was localized to the basolateral membrane of acinar 

cells, the apical and basolateral membranes of centroacinar 

cells, intra- and extralobular ducts, and main pancreatic duct 

cells (199). Localization of the pNBC1 variant to the basolateral 

membrane of pancreatic duct cells was further confirmed 

with a pNBC1-specific antibody (21). Satoh and coauthors 

(177), using kNBC1- and pNBC1-specific antibodies, reported 

that in human pancreatic ducts pNBC1 was expressed basolaterally 

and acinar cells were unstained, whereas in the rat 

pancreas acinar cells were stained basolaterally, and ductal 

cells expressed pNBC1 both apically and basolaterally. In 

addition, kNBC1 was localized apically in some ducts. Recently, 

in a similar study, Roussa and coauthors (169) found 

that pNBC1 was localized to the basolateral membrane of rat 

ductal and acinar cells and that pNBC1 and kNBC1 were 

localized together to the apical membrane of certain ductal 

cells. Moreover, these authors reported that in rat kidney 

pNBC1, in addition to kNBC1, was also localized to the 

basolateral membrane of S1 and S2 segments of the proximal 

tubule. It is clear that additional studies are needed to determine 

the reason for the varying results among different studies. 

The expression pattern of pNBC1 has also been characterized 

in corneal endothelium, duodenum, epididymis, vas deferens, 

and parotid and submandibular glands, where it is 

thought to mediate basolateral sodium bicarbonate influx (21, 

48, 89, 150, 170, 192, 193, 203). Species differences may 

determine differences shown for relative distribution of 

kNBC1 and pNBC1. For example, pNBC1 is expressed in 

various rat eye tissues including cornea, conjunctiva, lens, 

ciliary body, and retina, whereas the expression of kNBC1 is 

� 2� 



Human Gene Protein Name Function Stoichiometry Electrogenicity 

SLC4A1 AE1, band 3 Cl � /HCO 3 � exchange 1:1 Electroneutral 

SLC4A2 AE2 Cl � /HCO 3 � exchange 1:1 Electroneutral 

SLC4A3 AE3 Cl � /HCO 3 � exchange 1:1 Electroneutral 

SLC4A4 NBC1, NBCe1 Na � -HCO 3 � cotransport 2:1;3:1 Electrogenic 

SLC4A5 NBC4*, NBCe2 Na � -HCO 3 � cotransport 2:1;3:1 Electrogenic 

SLC4A7 NBC3, NBCn1 Na � -HCO 3 � cotransport 1:1 Electroneutral 

SLC4A8 NDCBE Na � -driven Cl � /HCO 3 � exchange 2:1:1 Electroneutral 

SLC4A9 AE4† Cl � /HCO 3 � exchange 1:1 Electroneutral 

Na � -HCO 3 � cotransport 1:1 Electroneutral 

SLC4A10 NCBE† Na � -driven Cl � /HCO 3 � exchange 2:1:1 Electroneutral 

Na � -HCO 3 � cotransport 1:1 Electroneutral 

Names used in the literature for SLC4 transporters are as follows: SLC4A1: eAE1, (band 3), kAE1; SLC4A2: AE2a,b1,b2,c1,c2; SLC4A3: bAE3, cAE3; 

SLC4A4: kNBC1 (NBC1a, NBCe1-A); pNBC1 (NBCe1-B, NBC1b, dNBC1, hhNBC, rb1NBC); rb2NBC (NBCel-C); SLC4A5: NBC4a, NBC4b, NBC4c 

(NBCe2-C), NBC4d, NBC4e, NBC4f; SLC4A7: NBC3 (NBCn1-A), NBCn1-B, NBCn1-C, NBCn1-D, NBCn1-E; SLC4A8: NDCBE, kNBC-3; SLC4A9: AE4a, 

AE4b; SLC4A10: rb1NCBE (NCBE-B), rb2NCBE, NCBE-A. *The NBC4c splice variant functions in mammalian cells as an electrogenic sodium bicarbonate 

cotransporter (176). The NBC4e splice variant has been reported to function in Xenopus laevis as an electroneutral sodium bicarbonate cotransporter (222). †The 

functional properties of these transporters is controversial (see text for details and references). The stoichiometry for SLC4A4 and SLC4A5 transporters reflects 

the HCO 3 � :Na � coupling ratio. For SLC4A8 and SLC4A10 transporters, the 2:1:1 stoichiometry reflects the HCO3 � :Na:Cl � coupling ratio. 


F583 

restricted to the conjunctiva (21). In the human eye, kNBC1 

and pNBC1 appear to be more evenly distributed (202). 

SLC4A5 (NBC4, NBCe2). NBC4 was originally cloned from 

a human cardiac cDNA library (156). Six human NBC4 

COOH-terminal splice variants (NBC4a–f: 1,137, 1,074, 

1,121, 1,040, 1,051, and 760 aa, respectively) (156, 157, 222) 

have been cloned. The NBC4c splice variant has been shown to 

mediate electrogenic sodium bicarbonate cotransport when 

expressed in mammalian mPCT cells (176) and X. laevis 

oocytes (210). NBC4e mediated electroneutral sodium bicarbonate 

cotransport when expressed in X. laevis oocytes (222). 

NBC4e was identical to NBC4c except for deletion of the last 

70 aa and 2 aa substitutions (H612Y and A699T) that are not 

present in the human genome database. If confirmed, NBC4e 

would be the first known example of a sodium bicarbonate 

cotransporter whose transport stoichiometry and electrogenic 

properties change as a result of a deletion/modification of 

specific residues. NBC4 transcripts are expressed in several 

organs and tissues (156). In rat hepatocytes, NBC4c protein has 

been localized to the basolateral (sinusoidal) plasma membrane, 

whereas intrahepatic cholangiocytes stain apically (3). 

In the kidney, NBC4c is also expressed on the apical membrane 

of uroepithelial cells lining the renal pelvis (3), where the 

cotransporter may play an important role in protecting the renal 

parenchyma from alterations in urine pH. Kristensen and 

colleagues (109) have recently reported that NBC4 potentially 

regulates the pH of sarcolemmal vesicles in skeletal muscle. 

SLC4A7 (NBC3, NBCn1). The electroneutral sodium bicarbonate 

cotransporter NBC3 was originally cloned from human 

skeletal muscle (155). Human NBC3 consists of 1,214 aa and 

is the first known stilbene-insensitive member of the SLC4 

family. Subsequently, the rat ortholog of NBC3, the electroneutral 

sodium bicarbonate cotransporter NBCn1, was cloned 

from rat aorta (37). NBC3 (114) and NBCn1 (211) are apparently 

glycosylated to a greater extent than NBC1 proteins. 

Three rat NBCn1 splice variants named NBCn1-(B–D) were 

also cloned from rat aorta (37). 

SLC4A7 proteins are expressed in several tissues. NBC3 

protein is expressed in rat renal connecting tubule (114) and in 

rat and rabbit renal collecting ducts in the apical membrane of 

�-intercalated cells and the basolateral membrane of �-inter- 





F584 � 2� 


calated cells (114, 161). NBCn1 was detected in intercalated 

cells in the medulla and, in addition, at the basolateral membrane 

of the rat medullary TAL and inner medullary collecting 

duct cells (151, 211). NBC3 was also highly expressed on the 

apical membrane of narrow cells in caput epididymis and light 

(clear) cells in corpus and cauda epididymis (158). The cotransporter 

apparently plays a role in pHi regulation in the outer 

medullary and inner medullary collecting ducts (113, 151, 

227). In addition, the cotransporter plays a role in basolateral 

bicarbonate transport in the medullary TAL (23, 82, 139), 

proximal duodenal villus cells (150), the basolateral domain of 

choroid plexus epithelial cells (152), and submandibular glands 

(58, 122). 

Cooper and colleagues (42) recently reported a deletion 

variant (NBCn1-E) in rat hippocampal neurons that lacks 123 

amino acids in the cytoplasmic NH2-terminal domain. The 

cotransporter may play an important role in regulating hippocampal 

neuronal activity. 

Na�-Driven Cl� � 

/HCO3 Exchangers 

SLC4A8 (NDCBE). The first Na�-driven Cl� � 

/HCO3 exchanger 

(NDCBE) was cloned by Romero and coauthors (167) 

from Drosophila and named Na�-driven anion exchanger 1 

(NDAE1). Its transport was blocked by stilbenedisulfonates 

and might not be strictly electroneutral. In addition, NDAE1 

was able to use OH� instead of bicarbonate. NDCBE (1,044 

aa) cloned from the human brain by Grichtchenko and colleagues 

(60) when expressed in X. laevis oocytes functions as 

aNa�-driven Cl� � 

/HCO3 exchanger, has a strict requirement 

� � 

for HCO3 , and is DIDS sensitive. Although CO2/HCO3 stimulates 

NDAE1-induced pHi changes, it is not clear whether 

� � 

NDAE1 mediates HCO3 transport per se because CO2/HCO3 could simply dissipate OH� in the adjacent unstirred layer. 

NDAE1 expressed in X. laevis oocytes also differs from human 

NDCBE in that it is associated with a Cl� current, as well as 

� 

an inward current caused by CO2/HCO3 . NDCBE transcripts 

are present in the brain, testis, kidney, and ovary (60). 

A splice variant of NDCBE (originally named kNBC-3) was 

cloned from mouse inner medullary collecting duct 

(mIMCD-3) cells and when expressed in X. laevis oocytes 

mediated electroneutral sodium bicarbonate cotransport (212). 

The chloride dependence was not studied, and therefore its 

functional properties remain unclear. More recently, Virkki 

and coauthors (209) reported the cloning of a Na�-driven Cl� � 

/HCO3 exchanger of �1,200 aa from squid giant fiber lobe 

(sqNDCBE). Unlike the transport process described in native 

squid axon (22), reversal of the Na� gradient alters the direction 

sqNDCBE transport, and Li� can support pHi recovery 

from an acid load nearly as well as Na� . 

SLC4 Transporters Whose Function is 

Incompletely Characterized 

SLC4A9 (AE4). AE4 was originally cloned (AE4a, 955 aa, 

and AE4b, 939 aa) from rabbit �-intercalated cells (202) and 

was localized to the apical membrane of renal collecting duct 

�-intercalated cell. Rabbit AE4a expressed in X. laevis oocytes 

and COS cells mediated Na � -independent, DIDS-insensitive 

Cl � /HCO 3 � exchange. In a separate study, AE4 was localized 

to apical and lateral membranes of collecting duct �-intercalated 

cells in the rabbit (105). However, in rat and mouse 


collecting ducts, AE4 was detected on the basolateral membrane 

of �-intercalated cells (105). AE4 is also expressed on 

the basolateral membrane of mouse submandibular gland duct 

(105). In contrast to the data of Tsuganezawa and colleagues 

(201), rat AE4 expressed in LLC-PK1 and HEK293 cells 

mediated DIDS-sensitive Cl � /HCO 3 � exchange (105). Parker 

and colleagues (144) characterized human AE4 (945 aa) expressed 

in X. laevis oocytes as an electroneutral sodium bicarbonate 

cotransporter without Cl � /HCO 3 � exchange activity. 

Whether these discrepancies arise from species-mediated differences 

in amino acid composition or expression systemspecific 

protein(s) potentially interacting with AE4 requires 

further study. Based on its amino acid sequence, AE4 is most 

homologous to electrogenic sodium bicarbonate cotransporters 

rather than AE1–3. 

SLC4A10 (NCBE). NCBE (1,088 aa) was originally cloned 

from a mouse insulinoma cell line and when expressed in X. 

laevis oocytes and HEK293 cells was reported to mediate 

Na � -driven Cl � /HCO 3 � exchange (213). Two splice variants 

have been subsequently cloned from rat brain and functionally 

expressed (57). rb1NCBE was similar to mouse NCBE except 

for a 30-aa insert in the NH2 terminus and was highly expressed 

in spinal cord and brain beginning in the embryo. 

rb2NCBE has an additional 18 aa in its COOH terminus, which 

ends with a PDZ motif. rb2NCBE is more highly expressed in 

astrocytes than rb1NCBE. Both transcripts mediated Na � - 

driven Cl � /HCO 3 � exchange when expressed in mouse fibroblast 

3T3 cells, but rb2NCBE was more active in pH regulation 

(57). A human ortholog of rb1NCBE, NCBE-B (1,118 aa), and 

its splice variant missing 30 aa in the NH2 terminus, NCBE-A, 

have been reported (39). 

NCBE transcripts have been detected in the peripheral nervous 

system and in nonneuronal tissues such as the choroid 

plexus, the dura, and some epithelia including the acid-secreting 

epithelium of the stomach and the duodenal epithelium (76) 

and kidney (153). rb1NCBE protein was highly expressed on 

the basolateral membrane of mouse and rat choroid plexus cells 

(152, 153). rb2NCBE protein has also been localized in preliminary 

studies to the basolateral membrane of mTAL intercalated 

cells in the inner medullary collecting duct and duodenal 

villus cells (153). In a preliminary study, Choi and coauthors 

(39) demonstrated that the human NCBE-B splice variant 

functions in X. laevis oocytes as an electroneutral Na � -HCO 3 � 

cotransporter. Whether experimental differences in the extent 

of Cl � depletion and/or expression systems account for these 

findings is currently unknown. 

SLC4 Transporters That Do Not Transport Bicarbonate 

SLC4A11 (NaBC1). SLC4A11 (initially called BTR1) (145) 

was originally included in the SLC4 family because of distant 

sequence homology to other family members. BTR1 has been 

renamed NaBC1 because it has been shown to function as a 

Na�-dependent borate transporter (143, 145). In the absence of 

borate, NaBC1 conducts Na� and OH� (H� ), whereas in the 

presence of borate, NaBC1 functions as a voltage-regulated, 

electrogenic Na� � 

-B(OH) 4 cotransporter with a stoichiometry 

of Na� � 

:B(OH) 4 �2. NaBC1 is the mammalian ortholog of the 

Arabidopsis thaliana borate transporter AtBor1 (194). 




CHANNEL-LIKE PROPERTIES OF SLC4 TRANSPORTERS 

Several electroneutral SLC4 transporters also have channellike 

properties. AE1 in native X. laevis oocytes has an associated 

small conductive anion current (51). The Na � -driven 

Cl � /HCO 3 � exchanger cloned from Drosophila NDAE1 also 

mediates a small Cl � current and a HCO 3 � current (167). Choi 

and colleagues (37, 42) have shown that two rat ortholog splice 

variants of the human NBC3 (NBCn1-B and NBCn1-E) in both 

X. laevis oocytes and HEK293 cells have an associated ion 

conductance that, as they suggested, is likely mediated by the 

cotransporter per se rather than an associated protein, although 

the latter possibility has not been definitely ruled out. The 

associated current was stimulated by DIDS, is not bicarbonate 

dependent, and Na � appeared to be the predominant ion 

responsible for the current. The physiological importance of 

these data, as well as potential implications to the transport 

mechanism, is unclear. 

TRANSPORT STOICHIOMETRY 

The ion transport stoichiometry of SLC4 transporters determines 

whether they are electroneutral or electrogenic (Table 1) 

(67, 112). The flux through electrogenic SLC4 transporters is 

affected by both the membrane potential and the activity of the 

transported ion species. AE1–3 proteins exchange equivalent 

amounts of Cl� for base and are electroneutral (6, 33). Sodium 

bicarbonate cotransporters mediate the transport of Na� and 

base with different stoichiometries per transport cycle. In 

NBC3 (NBCn1), the transport stoichiometry is 1:1, and, like 

AE1–3, this transport process is electroneutral (37, 155). 

NBC1 mediates the cotransport of one positive charge and 

either two or three negative charges per transport cycle (2, 30, 

62–64, 67, 68, 112, 166). NBC4 is also electrogenic and has 

been reported to have a 3:1 transport stoichiometry when 

expressed in mammalian cells (176) and a 2:1 stoichiometry in 

X. laevis oocytes (211). Human NDCBE exchanges Cl� for the 

equivalent of one Na� � 

and two HCO3 ions, and the process is 

electroneutral (60) (see Fig. 1 for other potential modes of 

transport). Although all studies agree that AE4 and NCBE are 

electroneutral transporters, their Cl� and Na� dependence are 

controversial (39, 57, 105, 144, 201, 213), and additional 

studies are required to determine their mode of transport. 

NBC1 and NBC4 transporters differ from other SLC4 family 

members in that they are electrogenic. The net charge carried 

by NBC1 and NBC4 varies with their transport stoichiometry 

and is an important determinant of the direction of Na� and 

� 2 

HCO3 (or CO3 

� 

) flux (Fig. 3) (67, 112). In addition, because 

the cell membrane potential induces changes in the flux of 

electrogenic sodium bicarbonate cotransporters, the effective 

cell buffer capacity will also vary with the membrane potential 

(16). This provides an interesting mechanism for coupling 

unrelated transport processes such as the electroneutral monocarboxylate 

transporter via MCT1 (which transports H� and 

moncarboxylates) to changes in membrane potential in cells 

expressing kNBC1 (16). 

It had been originally thought that the transport stoichiometry 

of SLC4 proteins is an inherent property of each transporter 

that remains fixed. However, Chernova and colleagues 

(35) showed that an E699Q mutation in mouse AE1 blocked 

electroneutral Cl� /Cl� anion exchange and created an electrogenic 

Cl� 2� 

/SO4 exchange process. These data indicated that a 

� 2� 



Fig. 3. HCO 3 � :Na � transport stoichiometry and ion gradients of the cotransported 

species determine the reversal potential (Erev) and therefore the direction 

of transport mediated by electrogenic SLC4 transporters (67, 112). 

Depicted is the effect of changes in either intracellular-to-extracellular Na � 

concentration ([Na � ]i/[Na � ]o; A) or intracellular-to-extracellular HCO 3 � concentration 

([HCO 3 � ]i/[ HCO3 � ]o ; B) ratio on Erev of an electrogenic Na � - 

HCO 3 � cotransporter functioning with a HCO3 � :Na � cotransport stoichiometry 

of either 2:1 or 3:1. The plot was generated using the following equation: 

E rev � RT 

F�n � 1� ln �Na� i 

�HCO 3 � � i n 

� n 

�Na�o �HCO3 � o 


F585 

where R, T, and F have their usual meaning, and n is the HCO 3 � :Na � transport 

stoichiometry. In A, [HCO 3 � ]i � 15 mM and [HCO3 � ]o � 25 mM. In B, 

[Na � ]i � 20 mM and [Na � ]o � 150 mM. When the membrane potential is 

more negative than the Erev, the cotransporter functions in the efflux mode, 

thereby contributing to transepithelial HCO 3 � absorption as in the renal 

proximal tubule. Although it is usually assumed that a cotransporter functioning 

in the 2:1 mode mediates HCO 3 � influx as in the pancreas, in the rabbit 

proximal tubule when the cotransporter functions in the 2:1 mode, at a 

basolateral membrane potential of about �50 mV, basolateral HCO 3 � efflux 

can be driven by a high [Na � ]i of �60 mM (175, 181). 

single amino acid substitution could shift an electroneutral 

transport process to an electrogenic one. In addition, following 

the cloning of the NH2-terminal variants of NBC1 (kNBC1 and 

pNBC1), it had been thought that kNBC1 functions in a 3:1 

(efflux) mode and pNBC1 functions in 2:1 (influx) mode. 

Gross and colleagues (63), however, showed that both transporters 

can function in either a 2:1 or 3:1 mode depending on 

the cell type in which they were exogenously expressed. In 

addition, it was shown that the HCO 3 � :Na � transport stoichiometry 

of NBC1 (kNBC1 and pNBC1) can shift from 3:1 to 

2:1 following cAMP-induced PKA-dependent phosphorylation 

of kNBC1-Ser 982 /pNBC1-Ser 1026 (62, 64). Moreover, Asp 986 

and Asp 988 were also shown to be required for the cAMPinduced 

shift of kNBC1 stoichiometry (68). These residues are 

located in close proximity to the PKA phosphorylation site, 

K 979 KGS, and are part of a putative D 986 NDD motif that, in 

addition to a second motif, L 958 DDV, was shown to be involved 

in carbonic anhydrase II (CAII) binding (154). 

Based on these considerations, Gross and colleagues (67, 68) 

originally hypothesized that a potential mechanism for the 

cAMP-induced shift in stoichiometry of kNBC1 requires the 

binding/dissociation of CAII. Against this hypothesis was the 

subsequent finding by Pushkin and colleagues (154) that the 

PKA-dependent phosphorylation of Ser 982 does not decrease 

CAII binding in vitro. In addition to PKA-dependent phosphorylation, 

an increase in intracellular Ca 2� concentration shifts 

the stoichiometry of kNBC1 expressed in X. laevis oocytes 

from 2:1 to 3:1 (132). The mechanism for the Ca 2� -induced 

shift in stoichiometry is currently unknown. Additional studies 

have shown that PKA-dependent phosphorylation of pNBC1 





F586 � 2� 


(KRKT 49 ) does not alter the transport stoichiometry but enhances 

flux through the cotransporter, thereby potentially playing 

an important role in secretin-induced stimulation of pancreatic 

bicarbonate secretion (62). 

TRANSPORTED BASE: HCO 3 � VS. CO3 2� 

It has not been definitively established whether SLC4 pro- 

� 2 

teins mediate HCO3 and/or CO3 

� 

transport. Depicted in Fig. 1 

are the hypothetical modes of transport wherein the net charge 

� 2 

per transport cycle by HCO3 and/or CO3 

� 

with Na� and/or 

Cl� is equivalent. Müller-Berger and colleagues (135) suggested 

that the finding that carbonic anhydrase (CA) inhibition 

only altered the flux through the basolateral proximal tubule 

electrogenic sodium bicarbonate cotransporter (kNBC1) functioning 

in the 3:1 mode and not in the 2:1 mode could be used 

� 2 

to determine whether HCO3 and/or CO3 

� 

was the transported 

species. These authors argued on theoretical grounds that when 

CA inhibitors decrease the flux through the cotransporter, 

2 

CO3 � 

must be one of the transported species and that, in the 3:1 

2 

mode, 1CO3 � � � �1HCO3 � 1Na are transported. Moreover, 

the finding that CA inhibition did not alter the flux in the 2:1 

2 

mode argued against CO3 � 

� � 

flux in favor of 2HCO3 � 1Na 

being transported. More recently, it has been suggested in 

preliminary experiments that even in the 2:1 mode, kNBC1 

transports 1Na� 2 

and1CO3 � 

(59). 

2 

Another potential approach to distinguish CO3 � 

� 

from HCO3 transport is the use of equilibrium thermodynamics (112). In 

the presence of a stable PCO2 difference across a membrane 

with a functional kNBC1 for example (e.g., lipid bilayer), it 

was previously demonstrated that theoretically under these 

conditions, the respective reversal potential values for a 

� � 2 

3HCO3 � 1Na transport mode vs. a1CO3 

� 

� 

� 1HCO3 � 

1Na� transport mode would differ. Another potential approach 

by which to distinguish these modes of transport would be to 

establish a stable pK difference across a membrane (112). This 

� 2 

is possible to accomplish because the HCO3 3 CO3 

� 

� H� reaction varies with ionic strength. There are, however, technical 

limitations that have thus far not been solved that have 

prevented these approaches from being used experimentally. 

Fig. 4. Phylogenetic tree of SLC4 transporters created using 

the UPGMA analysis. Numbers on top of each line represent 

the relative difference between the specific sequences using 

GeneWorks software. 

STRUCTURE 

Primary Structure 


Comparison of the primary structures of SLC4 transporters 

shows significant sequence homology especially in putative 

membrane domains (Fig. 2). On the basis of their amino acid 

sequence homology, SLC4 transporters appear to be separated 

structurally into three groups (Fig. 4) that likely have an 

evolutionary basis: 1) SLC4A1, SLC4A2, and SLC4A3; 2) 

SLC4A4, SLC4A5, and SLC4A9; and 3) SLC4A7, SLC4A8, 

and SLC4A10. These groups are distinct from the known 

functional categorization of SLC4 transporters, indicating that 

primary structure may not be a sufficient predictor of the 

functional properties of each transporter. 

Posttranslational Modifications 

Although all SLC4 transporters have various sites for potential 

posttranslational modification, thus far AE1 and NBC1 

transporters have been studied in some detail. Human erythrocyte 

AE1 contains a single site of N-glycosylation (Asn642 )in 

the fourth exofacial loop (Figs. 2 and 5) that contains either a 

short complex oligosaccharide or an extended polylactosaminyl 

oligosaccharide (33). Approximately equal amounts of the 

different glycosylated forms of AE1 are found in human red 

blood cells. Recently, a novel glycosylation site at Asn555 in 

the third exofacial loop has been described in addition (36). 

kNBC1 has been shown to contain two normally glycosylated 

sites at Asn597 and Asn617 (38) located in the second exofacial 

loop (Fig. 6) (198). Human AE1 was also palmitoylated at 

Cys843 (36). Interestingly, this modification did not affect 

trafficking of the anion exchanger. 

kNBC1 and pNBC1 are phosphorylated in their common 

COOH-terminal PKA phosphorylation site, KKGS (62, 64, 

68). The PKA-mediated phosphorylation of Ser982 in kNBC1 

(Ser1026 in pNBC1) shifts the transport stoichiometry of both 

NBC1 variants from 3:1 to 2:1 (62, 64, 68). pNBC1 possesses 

an additional PKA site (KRKT49 ), involved in the regulation of 

pNBC1-mediated flux but not the cotransporter stoichiometry 

(62). 




Topological Models 

Different algorithms predict 9–15 transmembrane segments 

in members of the SLC4 family. Topological models have been 

experimentally evaluated in two SLC4 transporters. The most 

extensively studied SLC4 transporter, the anion exchanger 

AE1, was proposed to possess 12 (149), 13 (33, 54, 232), or 14 

transmembrane segments (70, 94), of which the first 8 are 

generally accepted to exist (Fig. 5). The models fit calculations 

of AE1 transmembrane area based on a low-resolution twodimensional 

crystallography data of dimeric COOH-terminal 

membrane domain of AE1 (214). All AE1 models predict that 

the NH2 and COOH termini are localized intracellularly. Controversial 

experimental data regarding putative COOH-terminal 

transmembrane segments require use of high-resolution 

� 2� 



Fig. 5. Human AE1 topology model based on the work of Zhu and colleagues (232). Amino acids that are important for AE1-mediated transport are shown in 

red (acidic), blue (basic), and black (others). Green stars depict the residues involved in human disease. 

X-ray crystallography to precisely identify the number and 

location of AE1 transmembrane segments. 

Several studies indicated that the integrity of several cytoplasmic 

loops of AE1 is not essential for assembly into an 

anion transport-active structure. Coexpression of two pairs of 

complementary fragments of AE1 resulted in the generation of 

the stilbene disulfonate-sensitive uptake of Cl � into X. laevis 

oocytes, similar to expressed intact AE1 (69). For example, the 

pairs of fragments, which contained the first 8 and the last 6 

membrane spans, and the first 12 and the last 2 membrane 

spans, separated within cytoplasmic surface loops of the membrane 

domain, were able to generate stilbene disulfonatesensitive 

Cl � uptake. In contrast, the pairs separated in the 

second cytoplasmic loop were not active (216). Omission of 

Fig. 6. Human NBC1 (NBCe1) topological model (198). Amino acids that are important for NBC1-mediated transport are shown in red (acidic), blue (basic), 

and black (others). Green stars depict the residues involved in human disease. TM, transmembrane. 


F587 





F588 � 2� 


any of single transmembrane segments except of spans 6 and 7 

caused complete loss of functional expression (70). The unusually 

stable association between the AE1 fragments divided 

in the second exofacial loop was suggested to indicate an 

existence of interactions between polar surfaces on amphiphilic 

portions of the third and fifth transmembrane spans. Most AE1 

fragments contained the necessary information to fold in the 

membrane (71). 

Tatishchev and colleagues (198) demonstrated that NBC1 

has 10 transmembrane segments, with the NH2 and COOH 

termini localized intracellularly (Fig. 6). Whether the difference 

in the number of transmembrane segments in AE1 and 

NBC1 is related to their specific functional properties remains 

to be determined. 

Tertiary Structure 

The tertiary structure of the members of SLC4 family has 

only been partially determined. A low-resolution (20 Å) structure 

of the membrane domain of AE1 has been reported based 

on electron microscopy and three-dimensional image reconstruction 

of negatively stained two-dimensional crystals (215). 

The dimeric membrane domain revealed a U-shaped structure 

with dimensions of 60 � 110 and a thickness of 80 Å. The 

structure is open on the top and at the sides, with the monomers 

in close contact at the base, and the basal domain was 40 Å 

thick. The upper part of the dimer consisted of two elongated 

protrusions, which formed the sides of a canyon, enclosing a 

wide space that narrowed down and converged into a depression 

at the center of the dimer on the top of the basal domain. 

This depression might represent the opening to a transport 

channel located at the dimer interface. 

The crystal structure of the NH2-terminal cytoplasmic domain 

of the human AE1 (aa 1–379) has been reported at a 

much higher (2.6 Å) resolution (230). A tight symmetrical 

dimer stabilized by interlocked dimerization arms contributed 

by both monomers was reported. The dimer fit within a 

rectangular prism of approximate dimensions 75 � 55 � 45 Å. 

Each subunit also included a larger peripheral protein binding 

domain with an ��-fold. The binding sites of ankyrin, 

deoxyhemoglobin, aldolase, and glyceraldehyde-3-phosphate 

dehydrogenase were localized in the structure. 

The COOH-terminal cytoplasmic domain of human NBC3 

(aa 1127–1214) expressed in Escherichia coli cells and analyzed 

by gel permeation chromatography and sedimentation 

velocity centrifugation had a Stokes radius of 26 and 30 Å, 

respectively (119). Shape modeling suggested that the COOH 

terminus had a rodlike shape with the dimensions of 16 � 190 

Å, which was supposed to promote binding of regulatory 

proteins (119). 

Oligomeric Structure 

It is not known whether SLC4 transporters are functionally 

active in monomeric form or whether oligomerization is required 

for their activity. Numerous biochemical studies of 

mammalian AE1 have identified monomers, dimers, trimers, 

tetramers, and higher order oligomers and their mixtures (32, 

41, 50, 136). The relative abundance of these forms was 

dependent on the detergent used for isolation of AE1 and the 

temperature of isolation. In two-dimensional crystals of AE1 

(50), the protein subunits, each consisting of two AE1 mono- 

mers, were arranged with threefold symmetry, suggesting that 

a functionally active form of AE1 might be a dimer or/and even 

a hexamer. Based on these structural data and because a 

mixture of dimers and tetramers has been shown to represent 

the majority of AE1 in red blood cells (19, 32, 41, 50, 136, 141, 

215), it is widely accepted that a dimer is an active oligomeric 

form of AE1. On the basis of coimmunoprecipitation and 

functional expression in X. laevis oocytes of noncontiguous 

and overlapping pairs of membrane domains of human AE1, 

Groves and Tanner (70) hypothesized that transmembrane 

spans 1–5 and 9–12 are involved in, and spans 6–8, 13, and 14 

are not important for, dimerization of AE1. 

Dimers and to a lesser extent tetramers were also shown to 

be major components of bAE3 purified from rabbit renal 

microsomal membranes in the presence of Triton X-100 (160). 

Tetramers of bAE3 were further characterized by transmission 

electron microscopy of negatively stained ion-exchanger molecules 

(160). Image analysis of the tetramer micrographs 

revealed an anion exchanger molecule in which monomers 

were arranged with fourfold symmetry around a cavity in the 

center of the molecule, which could play a role of an iontransporting 

channel. Oligomers of human NBC1 and NBC3 

were also detected when these proteins were expressed in 

HEK293 cells (159). 

TRANSPORT MECHANISM 


It is unusual that the same mutigene family contains transport 

proteins that function as exchangers (e.g., AE1, AE2, 

AE3, NDCBE) and cotransporters (e.g., NBC1, NBC4) (Fig. 

1). AE1 exchanges equal amounts of Cl � for HCO 3 � by a 

ping-pong mechanism, wherein the transporter can be in either 

the inward-facing or the outward-facing state (52, 55, 72, 88). 

According to Jennings et al. (88), the AE1 catalytic cycle 

consists of one pair of exchanging anions per anion exchanger 

monomer. The transport rate is governed by a single AE1 

conformational change, which leads to the transfer of a single 

anion across the membrane (146). The dissociation/association 

of ions with AE1 occurs more quickly than the AE1 conformational 

changes leading to the translocation of the bound 

anion. Human AE1 has an intrinsic functional asymmetry; 

therefore, at near neutral pH and equal extra- and intracellular 

concentrations of Cl � , most of the anion exchanger molecules 

are in the inward-facing state (104). 

The stilbene inhibitor DIDS has been shown to bind preferentially 

to the outward-facing state of AE1 (88) and is inhibited 

by DIDS from outside (31, 96). In contrast to AE1, kNBC1 

was inhibited equally by intra- or extracellularly applied DIDS 

(74). In addition, H2DIDS has been shown to covalently bind 

Lys 539 and Lys 851 in human AE1 at physiological pH (140), 

whereas DIDS binds to Lys 851 only at pH �12 (93). According 

to current AE1 topological models, both lysine residues are 

located in transmembrane segments (54, 70, 111, 232). kNBC1 

(30, 166) and the pancreatic form of NBC1, pNBC1 (2), as 

well as all members the SLC4 family with the exception of the 

electroneutral sodium bicarbonate cotransporter NBC3 (155) 

and rabbit AE4a (202) are stilbene inhibitable, supporting an 

idea of a possible shared mechanism(s) of ion transport. However, 

against this assumption is the finding that stilbene inhibition 

is not limited to the members of SLC4 family, suggesting 

that the mechanism of this inhibition is nonspecific and 




involves binding of stilbene to exposed lysine residues, leading 

to steric blocking of normal ion transport mechanism. For 

example, various members of the SLC26 anion exchanger 

family, which have shared no sequence homology with SLC4 

transporters, are also stilbene sensitive (131). 

Kinetic studies of the electrogenic sodium bicarbonate cotransporter 

NBC1 suggest that contrary to AE1, binding of 

both sodium and bicarbonate to the cotransporter is required 

before a transport event (65, 90). Gross and Hopfer (66) 

developed a kinetic model in which bicarbonate and sodium 

bind to NBC1 in an ordered rather than a random manner. 

Using additional simplifying assumptions, they fitted the 

model to experimental current-voltage relationships obtained at 

different concentrations of Na � and HCO 3 � and predicted that 

binding of the ions to the cotransporter is voltage dependent, 

with an electrical coefficient of 0.2 at pH 7.5. The later 

suggested that HCO 3 � “senses” �20% of the membrane’s 

electric field on binding to the cotransporter or, in other words, 

that the binding site for HCO 3 � is located about one-fifth of the 

electrical distance into the membrane. Two basic amino acid 

residues, Lys 854 and His 907 , in transmembrane segments of 

kNBC1 (Fig. 6) (198) potentially involved in binding of 

bicarbonate (1) are located �20 and �10% of membrane width 

from the cytoplasmic side. Therefore, binding of bicarbonate to 

kNBC1 in the inward-facing conformation may fit the prediction 

of Gross and Hopfer (66). 

ION SELECTIVITY AND TRANSLOCATION 

In proteins that function as exchangers and cotransporters, 

there are residues usually located outside transmembrane segments 

mediating ion selectivity, and residues usually located 

within transmembrane segments that are involved in ion binding 

and translocation (ion binding/translocation site) (44, 172, 

220). In the absence of the NH2-terminal domain, the COOHterminal 

domain of AE1 (61) and AE3 (108) was able to 

mediate Cl � /HCO 3 � exchange, suggesting that the NH2-terminal 

domain is not necessary for ion exchange. Several amino 

acid residues have been identified that might be involved in ion 

selectivity and binding/translocation of AE1. Specifically, 

treatment of human red cell AE1 with Woodward’s reagent K 

labeled only glutamate but not aspartate residues (86) and 

inhibited Cl � /Br � exchange, suggesting that glutamate residue(s) 

is involved in AE1-mediated ion transport. Pretreatment 

with 4,4�-dinitrostilbene-2,2�-disulfonate (DNDS) protected 

AE1 from binding and inactivation by Woodward’s reagent K, 

suggesting that the stilbene-binding site and the glutamate 

residue(s) are located close to each other. In addition to 

glutamate, lysine, arginine, and histidine residues have been 

shown to be essential for AE1 transport activity (73, 84, 126, 

140, 146, 219, 228). 

The importance of glutamate residue(s) in AE1-mediated 

transport was further confirmed when Glu 681 in transmembrane 

segment 8 of human red cell AE1 (Glu 699 in mouse AE1) was 

shown to be involved in the ion exchange function of human 

(86, 87) and mouse AE1 (35). Mutation of Glu 681 in human 

AE1 and a homologous residue in human AE2 (182) to neutral 

or basic amino acids blocked transport of monovalent anions 

(Cl � and bicarbonate) but did not change maximum sulfate/ 

sulfate exchange. Interestingly, replacement of mouse AE1 

Glu 699 with aspartate blocked transport of both monovalent 

� 2� 




F589 

and divalent anions (133). Replacement of mouse AE1 Glu 699 

with glutamine inhibited Cl � /HCO 3 � exchange, but the mutant 

anion exchanger mediated an electrogenic Cl � /SO 4 2� exchange 

when expressed in X. laevis oocytes (35). It was suggested (87) 

that Glu 681 is the binding site for H � , which is transported with 

SO 4 2� during AE1-mediated H � -SO4 2� cotransport, and can be 

alternately exposed to the intracellular and extracellular media. 

In addition, interaction of mouse AE1 Glu 699 with His 752 was 

shown to be important for pH sensitivity of Cl � transport at 

low pH (133). Recently, it was shown that an additional 

glutamate residue is involved in Cl � binding and is part of a 

second Cl � binding/transport site in the AE1 monomer (85). 

In human kNBC1 (Fig. 6), Asp 754 (glutamate in AE1–3) 

flanks transmembrane segment 6 and plays a key role in 

kNBC1-mediated sodium bicarbonate cotransport, suggesting 

that the decreased length of aspartic acid compared with 

glutamic acid may be related to sodium dependence of ion 

transport (1). In addition, Asp 555 , Glu 559 , Asp 647 , Asp 685 , and 

Asp 778 are important for kNBC1 function (1). Asp 685 , conserved 

in Na � -dependent SLC4 transporters and AE4, is potentially 

involved in sodium binding/translocation (1). Asp 555 , 

conserved in all SLC4 transporters, is located in close proximity 

to Lys 559 , which has been suggested (1) to participate in 

bicarbonate binding and translocation. 

The involvement of mouse AE1 Lys 558 (Lys 539 in human 

AE1, Fig. 5) in AE1-mediated Cl � transport was hypothesized 

based on site-directed mutagenesis data (134). This amino acid 

residue located in a transmembrane span is also a target for 

covalent binding of H2DIDS and DIDS (93, 140). Both 

H2DIDS (DIDS at pH �12) and pyridoxal phosphate bind 

another lysine residue, Lys 851 (100, 140) and inhibit AE1. 

Several lysine residues (Lys 559 , Lys 667 , Lys 668 , Lys 681 , Lys 770 , 

Lys 771 , Lys 854 , and Lys 924 ) are functionally important for 

kNBC1 (1). Lys 854 is localized in transmembrane segment 8, 

which is conserved in Na � -dependent SLC4 transporters and 

potentially involved in ion binding/translocation mediated by 

NBC1. Human kNBC1 Lys 559 and Lys 924 are potential targets 

for DIDS inhibition. These residues are located extracellularly 

(Fig. 6); however, it is known that stilbenes inhibit kNBC1 

from both extra- and intracellular locations (74). Based on 

these data, Abuladze and coauthors (1) hypothesized that this 

part of the extracellular loop 2 in NBC1 is able to migrate 

between extra- and intracellular compartments. In addition, 

these authors suggested that this region is involved in bicarbonate 

binding in NBC1. The second half of this loop contains 

naturally glycosylated Asn 597 and Asn 617 (38) and therefore is 

located extracellularly. Lys 667 , Lys 668 , Lys 681 , Lys 770 , Lys 771 , 

and Lys 854 are probably involved in ion selectivity in NBC1. 

Four histidine residues located in transmembrane segments 

of mouse AE1, His 721 , His 752 , His 837 , and His 852 (His 703 , 

His 734 , His 819 , and His 834 in human AE1, Fig. 5) were shown 

by site-directed mutagenesis to participate in AE1-mediated 

ion exchange (133, 134). Mouse AE1 His 852 was shown to play 

a key role in the control of pH dependence of Cl � transport 

(133). It was suggested that the formation of a hydrogen bond 

between His 852 and Glu 699 (Glu 681 in human AE1) is essential 

for the decrease in Cl � transport at low pH. In addition, His 834 

in human AE1 was shown to play an essential role in the 

protein conformational changes during the ion exchange and is 

located in close proximity to the anion translocation site (92). 

It was also hypothesized that all these histidine residues inter- 





F590 � 2� 


act with mouse AE1 Lys 558 (Lys 539 human AE1), the target of 

covalent binding of DIDS (93, 140). All these histidine residues 

except His 819 are conserved in SLC4 transporters (Fig. 2). 

The only NBC1 homolog of these AE1 histidine residues 

located in transmembrane segments, kNBC1-His 907 (human 

AE1-His 834 ), was shown to be important for NBC1-mediated 

ion transport (1), suggesting that it can participate in bicarbonate 

binding/translocation. Human kNBC1-His 776 (His 734 in 

human AE1) located in the third intracellular loop is also 

important for kNBC1 function and apparently involved in 

bicarbonate selection (1), whereas the role of His 807 (His 734 in 

human AE1) has not yet been evaluated. 

Mutating Arg 509 and Arg 748 in mouse AE1 to lysine, threonine, 

and cysteine or lysine and glutamine, respectively, 

abolished Cl � /Cl � exchange activity in X. laevis oocytes (97). 

The Arg 734 mutant was supposed to be functionally inactive, 

whereas the Arg 509 mutant was possibly active but abnormally 

folded and therefore subsequently degraded. Because Arg 734 is 

conserved in AE1–3 and missing in Na � -dependent members 

of the SLC4 family, including AE4, this residue may be 

responsible for Cl � binding/translocation in anion exchangers. 

In addition, the kNBC1 Arg 538 , Arg 680 , Arg 722 , Arg 881 , and 

Arg 943 conserved in the SLC4 family are also important for 

cotransporter function (1, 75). 

Several uncharged amino acids were detected in kNBC1 

transmembrane segments (Ser 427 , Tyr 433 , Phe 656 , Ser 684 , and 

Ala 799 ) and in loops (Thr 485 , Ala 556 , Thr 671 , Thr 677 , and 

Thr 815 ) that are important for cotransporter function (1, 49, 75). 

Numerous uncharged amino acids in the membrane domain of 

AE1 have also been shown to be important for anion exchange. 

Tang and coauthors (195) mutated to cysteine the amino acids 

within and in close proximity to transmembrane segment 8. 

The cysteine mutants of this segment, Ala 666 , Ser 667 , Leu 669 , 

Leu 673 , Leu 677 , and Leu 680 , and in addition the intracellular 

mutants of Ile 684 and Ile 688 were inhibitable by sulfhydryl 

reagents. It was suggested that these amino acids could be a 

part of a AE1 translocation pore (195). Analysis of single 

cysteine mutants in the COOH-terminal area of human AE1 

(Phe 806 -Cys 885 ) in the last two putative transmembrane segments 

identified certain key residues in the Val 849 -Leu 863 

region (231). The putative ion selectivity filter was located at 

Ser 852 -Leu 857 (231). 

INTERACTION WITH CA: A TRANSPORT METABOLON 

CAs are a multigene family of enzymes that catalyze the 

reversible synthesis of bicarbonate from CO2 and H2O (24, 

180, 183, 200). Inhibition of CA activity affects SLC4 anion 

exchange and sodium bicarbonate cotransport function (28, 29, 

128, 174, 181, 186). Kifor and coauthors (102) on the basis of 

indirect evidence suggested first that red cell AE1 might 

interact with a cytosolic CA. Reithmeier and colleagues (163, 

206–208) demonstrated that the COOH terminus of AE1 binds 

CAII. Functional studies have demonstrated that CAII stimulates 

the transport function of AE1, AE2, and AE3 (45, 186). 

As a model for the CAII-mediated enhancement of HCO 3 � 

transport through AE1, it was hypothesized that a complex of 

AE1 and CAII functions in red blood cells as a transport 

metabolon. In this model, the efficiency of bicarbonate transport 

via AE1 is enhanced by the intermolecular transfer of 


bicarbonate between CAII and AE1, thereby eliminating the 

slower cytoplasmic diffusion of HCO 3 � between the two proteins 

(163, 186, 187, 206–208). In addition to CAII, CAIV was 

shown to bind AE1–3 (185). 

It is known that inhibition of CA activity in the renal 

proximal tubule of the kidney significantly decreases the rate of 

transepithelial bicarbonate absorption and basolateral sodium 

bicarbonate efflux (28, 29, 128, 174, 181). Complete inhibition 

of CA by 0.1 mM acetazolamide (ACTZ) decreased the flux 

through exogenously expressed kNBC1 by �65% when the 

transporter functioned with a HCO 3 � :Na � stoichiometry of 3:1 

(68) as in the in vivo proximal tubule (28) without altering its 

stoichiometry. ACTZ does not inhibit kNBC1-mediated flux 

when the cotransporter functions in the 2:1 stoichiometry mode 

(68, 181). 

The COOH terminus of kNBC1 (kNBC1-ct) was shown to 

bind CAII in vitro with high (Kd � 0.2 �M) affinity (68). Two 

kNBC1 acidic motifs, L 958 DDV and D 986 NDD, are involved in 

this binding (154). The complex of kNBC1 with CAII functioned 

as a transport metabolon (154). In pancreatic ducts, 

where pNBC1 mediates the basolateral influx of bicarbonate, 

CAII is highly expressed (137, 138). pNBC1 has a COOH 

terminus identical to kNBC1, and transfection of HEK cells 

with nonfunctional CAII has a dominant-negative effect on 

pNBC1 function (12), suggesting that pNBC1 also forms a 

functional complex with CAII. pNBC1 also binds CAIV (12). 

The COOH terminus of electroneutral sodium bicarbonate 

cotransporter NBC3, like NBC1 and AE1-AE3, binds CAII 

with high affinity (Kd �100 nM) (119). The human NBC3- 

D 1135 -D 1136 residues were essential for the interaction between 

the two proteins. In �-intercalated cells in the outer medullary 

collecting duct, the interaction between apical NBC3 and CAII 

may play an important role in pHi regulation. 

In addition to CA, certain SLC4 transporters interact with 

other cellular proteins. The cytoplasmic domain of AE1 contains 

sites for binding ankyrin, 4.1 and 4.2 proteins, glyceraldehyde-3-phosphate 

dehydrogenase, phosphofructokinase, deoxyhemoglobin, 

p72syk protein tyrosine kinase, and 

hemichromes (230) and functions as an anchoring site for these 

membrane-associated proteins. These interactions are important 

for regulation of cell flexibility and shape (121), glucose 

metabolism (120), ion transport (124), and cell life span (95). 

The AE1 NH2-terminal cytoplasmic domain is stabilized by 

interlocked dimerization arms contributed by both monomers 

(230). 

GENETIC DISEASES AND KNOCKOUT MODELS 

The crucial importance of SLC4 transporters in regulating 

intracellular pH and transporting bicarbonate in various cell 

types is best exemplified by the phenotype resulting from an 

abnormality in the functional properties of several members of 

the family (Table 2). 

SLC4A1 

Patients with AE1 mutations tend to have either hereditary 

spherocytosis (HS) or distal renal tubular acidosis (dRTA) but 

not both abnormalities. The reason for the lack of overlap 

between these syndromes is not understood. The majority of 

patients with HS-associated AE1 mutations have an autosomal 

dominant inheritance with missense mutations that are distrib- 




Table 2. Genetic diseases and mouse knockout models 

Gene Species Phenotype Inheritance Reference(s) 

SLC4A1 Human Anemia, spherocytosis, distal RTA, hypokalemia, 

hypercalciuria, nephrocalcinosis, nephrolithiasis 

uted throughout the transporter. The underlying abnormality 

thus far appears to be abnormal targeting to the plasma membrane 

due to misfolding of mutant transporters. In addition, a 

dominant-negative effect results in a decrease in wild-type (wt) 

AE1 membrane expression. Patients with autosomal dominant 

dRTA due to mutant AE1 have missense mutations in AE1- 

R589 (R589H, R589C, and R589S), S613F, A889X, G609R, 

and a truncation of 11 COOH-terminal residues, R901X (26, 

34, 83, 99, 171). The syndrome is characterized by abnormal 

urinary acidification, metabolic acidosis, nephrocalcinosis, hypercalciuria, 

and hypokalemia. Within a given family, the 

phenotypic expression can vary, likely because of background 

genes that are thus far unidentified (218). 

Studies of either red blood cells or heterologous expression 

systems demonstrated only modest changes in function that 

could not account for the urinary acidification abnormality in 

patients (26, 83). wt-AE1 in vivo and in polarized Madin- 

Darby canine kidney (MDCK) cells is targeted to the basolateral 

membrane. The predominant abnormality in the mutants 

characterized thus far in polarized MDCK cells (G609R and 

R901X) is the abnormal targeting of kAE1 to both the apical 

and basolateral membranes (47, 171). It is speculated that 

luminal bicarbonate secretion via mislocalized apical AE1 

decreases urinary acidification in these patients and that oligomerization 

of mutant AE1 with the wt-AE1 results in a 

dominant-negative effect by mistargeting wt-AE1 to the apical 

membrane. The magnitude and direction of apical AE1-mediated 

transport would be dependent on the respective cell/lumen 

HCO 3 � and Cl � gradients. Whether patients with abnormal 

luminal bicarbonate secretion can be distinguished by a higher 

than urinary PCO2 compared with patients with dRTA due to 

abnormal H � -ATPase function remains to be determined. 

Autosomal recessive dRTA is prevalent in Southeast Asia. 

Unlike patients with autosomal dominant dRTA, autosomal 

recessive dRTA presents earlier and tends to be of greater 

severity (14, 17, 98). These patients have been reported to have 

missense mutations in AE1 G701D, or various compound 

heterozygous combinations, including patients with G701D 

and the second allele having an in-frame AE1-400–408 deletion 

[called South Asian ovalocytosis (SAO)], (G701D/SAO), 

A858D/SAO, �V850/SAO, A858D/�V850, and G701D/ 

S773P (27, 197, 204, 226). The G701D mutant functions and 

is targeted normally in red blood cells but not in �-intercalated 

cells, which lack the targeting of glycophorin A (GPA) that is 

expressed in the red cell membrane (197). AE1 SAO is unable 

� 2� 


AD, AR 14,17,26,27,34,47,83,98, 

99,103,164,171,179, 

197,204,218,221,226 

Bovine Anemia, spherocytosis, distal RTA AD 80 

Mouse knockout Anemia, spherocytosis, runting AR 147,184 

SLC4A2 Mouse knockout Growth retardation, achlorhydria, edentulous, abnormal 

spermatogenesis, embryonically lethal 

AR 56,130 

SLC4A3 Human (polymorphism) Generalized seizures 173 

SLC4A4 Human Short stature, basal ganglia calcification, mental retardation, 

cataracts, band keratopathy, proximal RTA, hypokalemia 

AR 49,75,78,79,81,116,177,202 

SLC4A5 Human (polymorphism) Hypertension 15 

SLC4A7 Mouse knockout Retinal degeneration, mild auditory impairment AR 20 

RTA, renal tubular acidosis; AD, autosomal dominant; AR, autosomal recessive. 



F591 

to transport anions (179, 221). The A858D mutant functions 

abnormally, and its function is only partially rescued by GPA. 

In addition, the functioning of A858D is significantly decreased 

when coexpressed with �V850 or SAO (27). Similarly, 

the abnormal function of the �V850 mutant is not rescued 

completely by GPA (27). Recent detailed studies of the S773P 

mutant show that it is expressed at a low level, has a shorter 

half-life, is misfolded, and is targeted to the proteosome for 

degradation (103). 

Kittanakom and colleagues (103) have a proposed a model 

wherein AE1 mutants that cause autosomal dominant dRTA 

form heteroligomers with wt-AE1 that are retained in the 

endoplasmic reticulum, resulting in a dominant-negative effect. 

In contrast, AE1 mutants that cause autosomal recessive dRTA 

form homodimers which are targeted to the proteosome for 

degradation. Unlike mutants causing autosomal dominant 

dRTA, the S773P/wt-AE1 and G701D/wt-AE1 oligomers are 

targeted normally to the plasma membrane in HEK293 cells. 

Whether, as demonstrated with autosomal G609R and R901X 

mutations, homoligomers S773P and G701D homoligomers 

are targeted abnormally to the apical membrane in polarized 

cells remains to be determined. Interestingly, although patients 

with autosomal recessive dRTA typically have no red cell 

phenotype, patients with homozygous AE1-V488M have neonatal 

severe anemia and dRTA (164). Similarly, cattle homozygous 

for the AE1 R646X mutation have spherocytosis and 

dRTA (80). AE1 knockout mice have runting and severe 

anemia (147, 184). 


Although there are no known diseases in humans attributed 

to loss of AE2 function, targeted disruption of SLC4A2 in mice 

has recently been reported. Using a Cre/loxP strategy to disrupt 

expression of three of the four variants of AE2, Medina and 

coauthors (130) reported failure of spermiogenesis and infertility 

in male AE2�/� mice. Gawenis and colleagues (56) 

reported a more severe phenotype in AE2�/� mice related 

most likely to a different targeting strategy that resulted in the 

complete loss of AE2 expression. In this study, in some mice, 

loss of AE2 function was lethal to the embryo. In those 

AE2�/� mice that survived, most of them died around the time 

of weaning. The variability in survival suggests a role for 

background genes in modifying the severity of the phenotype. 

AE2�/� mice exhibited achlorhydria, moderate dilation of the 





F592 � 2� 


gastric gland lumens, and a reduction in the number of parietal 

cells. Ultrastructural analysis revealed abnormal parietal cell 

structure, with severely impaired development of secretory 

canaliculi and few tubulovesicles but normal apical microvilli. 

In addition, the mice had severe growth retardation and were 

edentulous, ataxic, and almost deaf. These studies demonstrate 

the requirement for normal AE2 function in various organ 

systems and, specifically, the requirement of AE2 for normal 

gastric acid secretion and for normal development of secretory 

canalicular and tubulovesicular membranes in mouse parietal 

cells. In humans, it is probable that that total loss of AE2 

function is embryonically lethal. 


No genetic disease has been described for AE3. A significantly 

increased 2600 C/A polymorphism variant (A867D 

substitution) was detected in patients with idiopathic generalized 

epilepsies (173). 


Igarashi and colleagues (78) first reported two unrelated 

patients with homozygous mutations in the SLC4A4 gene. The 

patients were mentally retarded, with short stature and severe 

proximal renal tubular acidosis (pRTA), systemic academia, 

and hypokalemia. Ocular abnormalities included glaucoma, 

cataracts, and band keratopathy. The first patient had a missense 

R298S mutation (kNBC1 numbering) whereas the second 

patient had a R510H substitution. These mutations are 

predicted to affect both the kNBC1 and pNBC1 variants of the 

NBC1 (SLC4A4) gene. The serum amylase was elevated, and 

thyroid abnormalities were documented. A head computed 

tomographic scan revealed bilateral calcification of the basal 

ganglia. In a subsequent patient reported by the same group 

with a Q29X nonsense mutation affecting only kNBC1, the 

patient had severe pRTA and glaucoma without band keratopathy 

or cataracts (79). Both kNBC1 and pNBC1 are expressed in 

the eye in a cell type-specific fashion. In the rat eye, Bok and 

colleagues (21) detected pNBC1 in the ciliary body, conjunctival 

surface cells, cornea, lens epithelium, and retina, whereas 

kNBC1 expression was restricted to conjunctival basal cells. 

Usui and colleagues (202) reported a more widespread expression 

pattern of kNBC1 in various cell types in the human eye. 

The finding that the patient with the Q29X mutation only had 

glaucoma might suggest that band keratopathy and cataracts 

are due to pNBC1 mutations, whereas glaucoma is due to loss 

of function of kNBC1. However, other causes of congenital 

pRTA and systemic acidemia such as Lowe’s syndrome (43) 

also cause glaucoma and cataracts in addition to mental retardation 

and growth abnormalities, indicating that one should be 

cautious at this point in attributing a particular ocular phenotype 

to loss of function of either NBC1 variant in patients with 

SLC4A4 mutations. Dinour and colleagues (49) reported a 

patient with a homozygous S427L SLC4A4 missense mutation. 

The patient had severe pRTA, glaucoma, corneal opacities, 

cataracts, and abnormal dentition. A computed tomographic 

scan showed no calcifications, and the patient was of normal 

intelligence. Inatomi et al. (81) also recently reported a patient 

with a homozygous deletion of A2311 (kNBC1 numbering), 

resulting in anomalous transcription of 29 amino acids followed 

by a stop codon. The patient had severe pRTA, renal 


insufficiency, elevated lipase and amylase levels, normal intelligence, 

glaucoma, corneal opacities, cataracts, tooth enamel 

defects, growth retardation, calcification of the basal ganglia 

and the left frontoparietal region, mild osteopenia, and normal 

thyroid function. 

Of the known NBC1 mutations causing pRTA in humans, 

several have been studied in heterologous expression system. 

The R298S and R510H missense mutations when assayed in 

ECV304 cells decreased the functional activity of kNBC1 by 

�55% (78). The same mutations in pNBC1 (R342S, R554H) 

also decreased the functional activity of pNBC1 by �50% 

(177). The S427L mutant that is targeted normally to the 

plasma membrane in X. laevis oocytes has significantly abnormal 

voltage and Na � -dependent transport (49). Interestingly, 

this mutant has no reversal potential accounting for the failure 

of kNBC1 to mediate sodium bicarbonate efflux in the kidney 

and eye. Horita and coauthors (75) recently identified three 

additional homozygous mutations (T485S, A799V, and 

R881C) in patients with severe pRTA and ocular abnormalities. 

Functional studies of these mutants in addition to the 

R298S and R510H mutants suggested that a 50% or greater 

reduction in kNBC1-mediated transport is required to cause 

severe pRTA (plasma HCO 3 � concentration � 13 mM). 

Li and coauthors (116) recently demonstrated that the kNBC1- 

R510H and S427L mutations not only have reduced intrinsic 

activity but are also mistargeted when expressed in polarized 

MDCK cells. The R510H mutant was predominantly retained in 

the cytoplasm, whereas the S427L mutant was mistargeted to the 

apical membrane. They also identified a kNBC1 COOH-terminal 

motif, QQPFLS (aa 1010–1015), is required for targeting of 

NBC1 to the basolateral membrane in MDCK cells (117). Within 

this motif, a kNBC1-F1013A mutant was abnormally targeted to 

the apical membrane. In a large-scale mutagenesis screen, Abuladze 

and coauthors (1) have shown that residues in several loops 

and transmembrane segments in kNBC1 are required for normal 

plasma membrane trageting. Studies of the kNBC1-A2311 deletion 

mutant failed to show any plasma membrane expression and 

functional activity in X. laevis oocytes (81). 

Recently, it has been suggested that the phenotype in patients 

with rod and cone degeneration (RP17 form of retinitis pigmentosa) 

due to missense mutations in CAIV results from an impaired 

wt-NBC1/CAIV transport metabolon in the endothelium of the 

choriocapillaris (224). The biologically active CAIV-R14W mutant 

did not stimulate NBC1 activity by failing to bind to NBC1, 

whereas the CAIV-R219S mutant did not stimulate NBC1 activity 

due to its enzymatic inactivity. Additional studies are needed to 

determine whether patients with NBC1 mutations have degenerated 

rods/cones and whether the ocular phenotype resulting from 

mutant CAIV can occur in the absence of functioning NBC1. In 

a separate study, Rebello and coauthors (162) have reported a 

different underlying mechanism for the RP17 form of retinitis 

pigmentosa. In patients with the CAIV-R14W mutation, their 

finding suggests that the mutant enzyme in endothelial cells in the 

choriocapillaris leads to ER stress due to an accumulation of 

unfolded protein, apoptosis, and ischemia of the retina. 


Polymorphisms in this gene are associated with hypertension 

(15). Further studies are needed to clarify the basis for this 

association. 





Recently, the importance of NBC3 in sensory transduction 

has been demonstrated in an SLC4A7 knockout mouse (20). 

SLC4A7 knockout mice have a slow degeneration and ultimate 

loss of photoreceptors in the eye and a mild hearing defect due 

to the loss of inner and outer hair cells in the inner ear. These 

findings indicate that SLC4A7 knockout mice are a model for 

Usher syndrome, a group of diseases characterized by visual 

loss and auditory impairment in humans (101). 

CONCLUSION 

The SLC4 family contains a functionally diverse group of 

transport proteins that play an essential role in the transport of 

� 2 

base (HCO3 ,CO3 

� 

) in various tissues in mammals. Although 

there are have been numerous studies of the structure and 

function of the initial member of the SLC4 family, AE1, more 

recently, additional members of the SLC4 family have been 

investigated. Because of the efforts of several laboratories, 

various heterogeneous diseases can now be attributed to members 

of the SLC4 family. Nevertheless, although much progress 

has been made, a thorough understanding of the structure and 

function of both wild-type and mutated transporters at a molecular 

level is lacking. The current topological models of AE1 

are still controversial because of the various methodologies 

used by different investigators to address this question. Because 

of these complexities, one cannot satisfactorily determine 

whether the larger number of transmembrane regions in 

AE1 compared with NBC1 represents differences in their ion 

binding sites/mechanism of transport. Nevertheless, recent 

studies on specific amino acid residues critical for NBC1mediated 

transport provide a structural basis for the molecular 

comparison of NBC1 and AE1. Results obtained thus far 

suggest that the molecular architecture and the transport mechanisms 

of NBC1 and AE1 have certain similar features, and at 

the same time, there are unique differences that might account 

for the separate transport modes (exchanger vs. cotransporter). 

In the future, successful crystallization of SLC4 proteins will 

contribute to an understanding of their transport mechanism 

and help address these and other currently unresolved questions. 

GRANTS 

This work was supported by National Institutes of Health (NIH) Grants 

DK-63125, DK-58563, DK-07789, and ES-012935-A1, the Max Factor Family 

Foundation, the Richard and Hinda Rosenthal Foundation, the Fredrika 

Taubitz Fund, National Kidney Foundation of Southern California Grant 

J891002, and American Heart Association (Western State Affiliate) Grant 

0365022Y. 

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