Critical Review
Bicarbonate Transport in Health and Disease
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada
Abstract
Bicarbonate (HCO32) has a central place in human physiology
as the waste product of mitochondrial energy production and
for its role in pH buffering throughout the body. Because
bicarbonate is impermeable to membranes, bicarbonate trans-
port proteins are necessary to enable control of bicarbonate
levels across membranes. In humans, 14 bicarbonate transport
proteins, members of the SLC4 and SLC26 families, function
by differing transport mechanisms. In addition, some anion
Keywords: bicarbonate transport; SLC26; SLC4; pH regulation; cell
volume; carbon dioxide; bicarbonate
Introduction
Bicarbonate Metabolism in Mammals
Carbon dioxide, the principal waste product of mammalian
energy production, is the product of mitochondrial pyruvate
dehydrogenase catalyzing oxidation of pyruvate to acetyl CoA
and CO2. Other enzymes metabolize carbon dioxide, most
importantly carbonic anhydrases (CAs; ref. 1), which facilitate
the reversible reaction: CO2 ! HCO32 1 H1, a reaction that
also occurs spontaneously at a slower rate.
Bicarbonate is a labile molecule, undergoing pH-
dependent coupled conversions (Fig. 1). Key to understanding
the role of HCO32 transport in mammals is that CO2 is a conju-
gate acid, forming carbonic acid in the presence of H2O (cata-
lyzed by CA enzymes), whereas HCO32 is a base (Fig. 1). CO2
freely diffuses across lipid bilayers. Whether CO2 diffusion
across the lipid portion of biological membranes is sufficient to
explain observed rates of CO2 movement in tissues is a matter
C 2014 International Union of Biochemistry and Molecular Biology
V
Volume 66, Number 9, September 2014, Pages 596–615
Address correspondence to: Joseph R. Casey, Department of Biochemis-
try, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Tel: 1780-
492-7203. Fax: 1780-492-0886.
E-mail: joe.casey@ualberta.ca
Received 9 July 2014; Accepted 10 September 2014
DOI 10.1002/iub.1315
Published online 1 October 2014 in Wiley Online Library
(wileyonlinelibrary.com)
596
Kumari Alka
Joseph R. Casey*
channels and ZIP metal transporters contribute to bicarbonate
movement across membranes. Defective bicarbonate transport
leads to diseases, including systemic acidosis, brain dysfunc-
tion, kidney stones, and hypertension. Altered expression lev-
els of bicarbonate transporters in patients with breast, colon,
and lung cancer suggest an important role of these transport-
ers in cancer. V
C 2014 IUBMB Life, 66(9):596–615, 2014
of debate (2,3). Strong evidence does, however, indicate that
aquaporin-1 and the red cell Rh proteins facilitate transmem-
brane CO2 flux (3). In contrast to CO2, HCO32 is unable to dif-
fuse across lipid bilayers, requiring integral membrane bicar-
bonate transport proteins to cross membranes. CO2/HCO32
equilibrium forms the principal pH buffering system, both
inside and outside cells. The pKa of the CO2/HCO32 equilibrium
(6.4) favors the accumulation of similar amounts of both the
membrane-permeant CO2 and membrane-impermeant HCO32
at equilibrium in blood (pH 7.2).
Bicarbonate Transport Overview
The focus of this review is the function, physiological, and
pathophysiological roles of bicarbonate transport proteins. As
HCO32 is a base, transport across membranes affects pH. The
addition of HCO32 increases pH (alkalinizes), and HCO32
removal reduces pH (acidifies). As will be discussed, bicarbon-
ate transporters thus have important roles in pH homeostasis
and acid/base secretion. In addition, bicarbonate transporters
contribute to removal of respiratory CO2 and are involved in
cell volume regulation. Stringent control of the HCO32 move-
ment across cellular membranes is thus critical.
Here, the focus is bicarbonate transporters, the class of
proteins undergoing a conformational change as they move
HCO32. In addition to bicarbonate transporters, anion chan-
nels and metal transporters can move bicarbonate, as dis-
cussed below.
Human Genome Organization categorizes Solute Carriers
(SLC) transporting substances across the plasma membrane as
SLC proteins (4). Fourteen gene products, SLC4A1–5, SLC4A7–
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Bicarbonate is labile. Bicarbonate (HCO32) is in pH-
FIG 1 driven chemical equilibria with carbonate (CO322)
and carbonic acid (H2CO3). Carbonic acid can be con-
verted to water and CO2 by the enzyme carbonic
anhydrase (CA). Finally, at air interfaces, as occurs in
the lungs, dissolved CO2 is in equilibrium with gase-
ous CO2.
10, SLC26A3, 4, 6, 7, and 9, facilitate bicarbonate transport in
humans (Table 1). The genes responsible for the bicarbonate
transport in humans, the preferred names of the transport
proteins, tissue distribution/location, and basic mechanism of
transport are summarized in Table 1. Phylogenetic analysis of
amino acid sequences of bicarbonate transporters reveals
these as forming the SLC4 and SLC26 families with distinct
evolutionary origins (Fig. 2).
We will discuss the function of each mammalian bicarbon-
ate transporter, followed by their contribution to normal physi-
ology and in disease. Finally, the review concludes with
broader discussions of the interactions of CAs with bicarbonate
transporters and a separate section on the emerging area of
the role of bicarbonate transporters in cancer.
Bicarbonate Movement Through Ion Channels
Anion channels have variable ability to select substrates and
some have permeability to HCO32. The cystic fibrosis gene
product (CFTR; ref. 58), the Ca11-activated anion channels
bestrophin (59) and Anoctamin 1 (60), and finally, the GABA
and glycine receptors/anion channels (61) all display HCO32
permeability. The cell’s negative membrane potential will
favor HCO32 efflux through these ion channels.
Although cystic fibrosis was originally thought to arise
from a Cl2 channel defect, in the recent years, defects in
HCO32 homeostasis has gained prominence as an important
part of the pathology (62). Reduced HCO32 in airway surface
liquid of patients with CF compromises mucus secretion (63)
and increases susceptibility to bacterial colonization (64). Fur-
thermore, patients with CF have defects in pancreatic secre-
tion. Pancreatic duct cells illustrate the role of CFTR in HCO32
secretion (Fig. 3; ref. 65).
Characterization of bestrophin 2-deficient mice suggests
that bestrophin 2 carries the cholinergically stimulated HCO32
flux in goblet cells of the colon (59). In this model of colonic
HCO32 secretion, bestrophin 2 at the basolateral surface loads
the cells with HCO32, which is then secreted into the colonic
lumen by apical SLC26 proteins (59).
Bicarbonate Transport by Metal Transporters
ZIP8 (SLC39A8) and ZIP14 (SLC39A14) are electroneutral diva-
lent metal/HCO32 symporters (66). ZIP8 and 14 localization to
apical membrane of kidney and intestine led to a suggestion
that they are involved in metal uptake (66). Substrates of these
ZIPs (Fe11, Zn11, and Cd11) are found physiologically at lev-
Alka and Casey
els much lower than HCO32, suggesting that ZIP activity will
have little effect on cellular or blood HCO32 levels. Conversely,
ZIP function will be sensitive to HCO32 concentration, provid-
ing a possible point of crosstalk between metal toxicity and
HCO32 homeostasis.
The SLC4 Family of Bicarbonate Transporters
The SLC4 gene family divides functionally into three subfami-
lies: electroneutral Na1-independent Cl2/HCO32 exchangers
(AE1-3), electrogenic Na1-coupled HCO32 cotransporters
(NBCe1 and NBCe2), and electroneutral Na1-coupled HCO32
cotransporters (NBCn1, NDCBE, NBCn2, and SLC4A9).
SLC4A11 clusters separately from other SLC4 members (Fig.
2) and has never been found to act as a bicarbonate trans-
porter. Rather, one report suggested the protein to act as a
borate transporter (67), or more recently, as a water translo-
cation pathway (68) or Na1/H1 exchanger (69).
Na1-Independent Cl2/HCO32 Exchangers of the SLC4
Family
AE1 (SLC4A1). Anion Exchanger 1 (AE1) was the first Cl 2/
2
HCO3 exchanger to be cloned (5). Related Cl2/HCO32
exchangers, AE2 and AE3, were identified shortly thereafter
(7,10). AE1–3 share similar organization into domains (below)
and facilitate gradient-driven electroneutral exchange of Cl2
for HCO32 across the plasma membrane. Erythrocyte AE1,
also called band 3 (for its position on Coomassie blue-stained
acrylamide gels of red cell membranes; ref. 70), is enormously
abundant, constituting 50% of integral red cell membrane pro-
tein. AE1 has a key role in enhancing the blood’s CO2 carrying
capacity. In body tissues, metabolically produced CO2 diffuses
from plasma across the red cell membrane, where cytosolic
CAII catalyzes conversion to HCO32 and H1. This H1 is called
the Bohr H1 and has the important role of binding hemoglo-
bin, reducing its affinity for O2. Influx of CO2 and catalysis by
CAII would very rapidly cease because of substrate accumula-
tion, were it not for AE1 action in movement of HCO32 into the
plasma, in exchange for Cl2. In the lungs, the cycle reverses,
driven by the drop in pCO2, and CO2 is exhaled to the atmos-
phere. The cytosolic C-terminus of AE1 binds cytoplasmic CAII
to form a “bicarbonate transport metabolon” (71), discussed
further below.
The AE1 membrane domain has roles other than bicar-
bonate transport. AE1 also facilitates HS2/Cl2 exchange,
thereby contributing to the metabolism and transport of the
vasodilator, H2S (72). Cysteine residues within the AE1 N-
terminal cytoplasmic domain bind NO, acting as a membrane
proximal reservoir for this vasodilator to be released on pas-
sage into capillaries (73). AE1 in red blood cells forms the
Diego blood group antigen, of which there are 50 alleles
(74,75). Finally, AE1 associates with the single transmembrane
segment protein, glycophorin A, and this complex forms the
Wrb blood group antigen (76).
The AE1 N-terminal cytoplasmic domain functions as an
organizational center for erythrocytes, controlling cell shape
and flexibility (77), regulation of glucose metabolism (78), and
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Human bicarbonate transport proteins

TABLE 1

Transport Gene (Other Tissue distribution Transport mechanism

protein names used) (Epithelial location) (Electrogenicity) References
2 2
AE1 SLC4A1 (Band 3) Kidney (kAE1), RBC (eAE1), Cl /HCO3 exchange 5, 6
and heart (Basolateral) (Electroneutral)
AE2 SLC4A2 Widespread (Basolateral) Cl2/HCO32 exchange 7–9
(Electroneutral)
AE3 SLC4A3 Heart, brain, retina, pituitary, Cl2/HCO32 exchange 10–12
adrenal gland, kidney, (Electroneutral)
and gastrointestinal tract
(Nonepithelial)
NBCe1 SLC4A4 (NBC1, pNBC, Kidney, heart, prostate, Na1-coupled HCO32 13–16
HNBC1, and hhNBC) colon, pancreas, stomach, cotransport (Electrogenic)
thyroid, and brain (Basolateral)
NBCe2 SLC4A5 (NBC4) Brain, epididymis, liver, spleen, Na1-coupled HCO32 17–19
cardiac muscle, smooth muscle, cotransport (Electrogenic)
kidney, spleen, and choroid
plexus (Apical)
NBCn1 SLC4A7 (NBC2 Skeletal muscle, smooth muscle, Na1-coupled HCO32 20, 21
and NBC3) heart, kidney, spleen, liver, cotransport (Electroneutral)
lungs, submandibular gland,
pancreas, and stomach
(Basolateral)
NDCBE SLC4A8 (NBC3) Brain, cardiac myocytes, testis, Na1-coupled Cl2/HCO32 22–26
oocytes, and kidney (Basolateral) exchange (Electroneutral)
SLC4A9 SLC4A9 (AE4) Kidney (Apical) Na1/HCO32 cotransport 27–29
(Electroneutral)
NBCn2 SLC4A10 (NCBE) Neurons, kidney, uterus, Na1-coupled Cl2/HCO32 30–32
adrenal cortex, choroid exchange or HCO32-dependent
plexus, and cardiac myocytes Cl2/Cl2 exchange (Electroneutral)
(Basolateral)
SLC26A3 SLC26A3 (PDS, DRA, Ileum, colon, cardiac myocytes, Cl2/HCO32 exchange, 33–38
and CLD) eccrine sweat gland, enterocytes, also SO422 (Electroneutral)
and epididymis (Apical)
SLC26A4 SLC26A4 (Pendrin) Thyroid, inner ear, kidney, Cl2/HCO32 exchange, also I2, 39–44
prostate, and airway epithelial formate, NO32, and SCN2
cells (Apical) (Electroneutral)
SLC26A6 SLC26A6 (PAT-1 Kidney, heart, pancreas, Cl2/HCO32 exchange, also 45–49
and CFEX) bronchial epithelium, prostate, oxalate, NO32, OH2, formate,
thymus, and intestine (Apical) and SO422 (Electroneutral)
SLC26A7 SLC26A7 Thyroid, type A intercalated cells, Cl2/HCO32 exchange, also 50–52
stomach, retina, and olfactory oxalate and SO42 Cl2 channel
epithelium (Basolateral) activity (Electrogenic)
SLC26A9 SLC26A9 Brain, salivary gland, heart, Cl2/HCO32 exchange, Cl2 channel, 50, 53–57
stomach, trachea, kidney, Cl2-independent HCO32 transport,
lung bronchiolar, and alveolar and NaCl cotransporter (Electrogenic)
epithelia (Apical)

598 Bicarbonate Transporters in Health and Disease

 The AE1 membrane domain has undergone intensive
investigation. Several different topology models for AE1 mem-
brane domain present the locations of AE1 amino acids rela-
tive to the plane of the lipid bilayer. One such model was
developed by structural homology modeling (see below; Fig. 5).
The role of Glu681 in anion exchange kinetics is established,
and this residue may be located at the permeability barrier of
AE1 (90,91). Substituted cysteine accessibility method (SCAM)
studies revealed Met664-Gln683 as forming transmembrane
segment 8 of human AE1, whereas Ser643-Met663 and Ile684-
Ser690 localized at extracellular and intracellular sites of the
transmembrane segment, respectively (88,92). Furthermore,
Arg589-Met656 regions formed a “vestibule” that leads anions
to the transport channel. Glu681 localized three amino acids
from C-terminus of the transmembrane segment 8, placing the
membrane permeability barrier within 5 Å of the intracellular
surface (93). One proposed topology model of human AE1 sug-
gested this domain to be composed of 13 transmembrane seg-
ments, and the region from Asp807-His834 lacked the typical
a-helical conformation (94). The proposed topology of the last

Phylogenetic analysis of human bicarbonate trans-

FIG 2 port proteins. Unrooted phylogenetic dendrogram
was constructed by ClustalW software (http://www.
genome.jp/tools/clustalw/), using human amino acid
sequences for the indicated proteins. Line lengths
indicate evolutionary distance. SLC4 and SLC26 rep-
resent two distinct families of bicarbonate. Functions
of these proteins cluster with evolutionary origin,
with SLC4 electroneutral Cl2/HCO32 exchangers (yel-
low), Na1-coupled bicarbonate transporters (red),
and SLC26 Cl2/HCO32 exchangers (magenta).
SLC4A11 has not been reported to transport bicar-
bonate. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]

life span of cells (79), arising from the domain’s ability to asso-
ciate with the cytoskeleton and glycolytic enzymes (80–82).
In addition to erythrocyte AE1 (eAE1), kAE1 (an N-
terminal truncated version of eAE1 transcribed from an alter-
nate promoter lacking the first 65 amino acids) is expressed in
the basolateral membrane of a-intercalated cells of the renal
collecting duct (Fig. 4; refs. 6 and 83). In the basolateral mem-
brane, kAE1 transports HCO32 from the cytosol to the blood,
thus contributing to renal acid secretion.
AE1 Structure. High-sequence identity between membrane
domains of AE1 and other members of the SLC4 family (30–
45% identity and 37–54% similarity) suggests that SLC4 mem-
brane domains share a common folded structure. Thus, AE1
provides the best insight into the structure of all SLC4 proteins.
Studies of AE1 topology and cryo-electron microscopy have
provided low-resolution structures for the membrane domain
(84–87). Human AE1 is composed of two domains: an N-
terminal cytoplasmic domain and a C-terminal membrane
domain with a short C-terminal cytoplasmic region.
Pancreatic bicarbonate secretion. Pancreatic fluid has
FIG 3 the highest HCO32 concentration of any bodily fluid,
about 140 mM (65). To achieve this transepithelial
flux, the cell is loaded with HCO32 by NBCe1 and by
diffusion of CO2 into the cell, followed by CAII-
mediated catalysis to HCO32 and H1. NHE1 removes
H1 and loads the cell with additional Na1. Patients
with cystic fibrosis have defective pancreatic HCO32
secretion, indicating a key role of CFTR in pancreatic
HCO32 secretion. The role of CFTR is twofold: 1)
CFTR is a Cl2 channel, providing extracellular Cl2 to
sustain Cl2/HCO32 exchange by SLC26A6, and 2)
CFTR is a HCO32 channel, mediating HCO32 efflux.
Not shown in this simplified model are the ion chan-
nels/Na1/K1-ATPase required to maintain homeosta-
sis. NBCn1 localizes to the apical membrane, but
functions in HCO32 salvage, not secretion (65). The
mechanism of pancreatic duct cell HCO32 secretion
has been thoroughly reviewed (65).

Alka and Casey 599


Bicarbonate transporters in the distal nephron. Two
FIG 4 classes of cells are critical to acid–base homeostasis in
the renal collecting duct: type A (acid secreting) and
type B (base secreting), which are part of the tubule
epithelium, connected together by tight junctions
(black oval between the cells). Basolateral SLC26A7
has also been identified in type A cells. Acid secretion
in type A cells is initiated by diffusion of the conjugate
acid, CO2 from blood into the cell. Carbonic anhydrase
II (CAII) converts CO2 to HCO32 and H1, loading the
cell with acid. Basolateral kidney AE1 (kAE1) removes
the HCO32 in exchange for Cl2, allowing sustained
catalysis by CAII. At the apical surface, H1-ATPase
secretes acid into the kidney tubule. Base secretion in
type B cells is initiated by CO2 diffusion from tubule
lumen to cytosol, where it is converted to HCO32 and
H1 by CAII. The process is sustained by SLC26A4-
mediated Cl2/HCO32 exchange. At the basolateral sur-
face, H1-ATPase pumps H1 back into the blood.
Topology model for AE1 on the basis of ClC homology. Topology model for human AE1 (88) developed by structural homology
FIG 5 modeling to the ClC crystal structure (89). Boxes with letters represent the structural elements designated in the ClC crystal
structure. Branched structure represents the position of N-linked glycosylation at N642. Numbers represent the amino acid
numbers. N and C termini are labeled, and the missing N-terminal cytoplasmic domain is marked by dots.
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two transmembrane segments describes this region as a signif-
icant portion of the ion translocation pathway. This region was
proposed to be part of the transmembrane pore lining, with an
important role in ion translocation (92,95). Furthermore, the
loop connecting the last two TMs was identified and shown to
be required for selection of substrate ions on the basis of
charge.
Recently, a 7.5-16Å resolution electron microscopy struc-
ture of the dimeric membrane domain of AE1 proposed struc-
tural similarity of this domain with CIC chloride channel
(84,96). Taking the X-ray structure of the CIC chloride channel
(89) as template, a three-dimensional AE1 homology model
developed was consistent with established biochemical con-
straints and residues corresponding to CIC transport mecha-
nism (88). Mutation of AE1 residues corresponding to ClC cata-
lytic residues significantly affected the AE1 function (88),
suggesting structural homology in the positioning of catalytic
residues. The AE1 homology model (Fig. 5) is consistent with
most data arising from SCAM experiments (88).
A homology model for the AE1 membrane domain devel-
oped, based on the bacterial uracil-proton symporter (UraA),
consists of 14 transmembrane segments and provides further
support for the role of TM3, 4, 5, and 8 in anion exchange
and cation leak (97). Although the UraA and the ClC-based
models for AE1 differ in structure, both feature an internal
duplication with twofold pseudosymmetric orientation
(89,98). Ultimately, a high-resolution structure is required to
establish AE1 structure and its relationship to transport
mechanism.
Bicarbonate Transporters in Health and Disease
 The crystal structure of the N-terminal cytoplasmic
domain of AE1 at 2.6 Å reported the protein to be a dimer sta-
bilized by interlocking dimerization arms contributed by each
monomer (99). Each subunit serves as a binding site for
peripheral proteins, including protein 4.1, ankyrin, hemoglo-
bin, and glycolytic enzymes.
AE1 in Disease. kAE1, expressed on the basolateral mem-
brane of acid secreting (also called type A, or a) intercalated
cells of the distal nephron, transports HCO32 from the cytosol
to blood as a part of the system of acid secretion into urine
(Fig. 4). Although most HCO32 reabsorption occurs in the
proximal tubule (see NBCe1 below), about 10% happens in a-
intercalated cells. kAE1 mediates Cl2/HCO32 exchange, trans-
porting HCO32 from the a-intercalated cell cytosol to blood.
Failure of this process causes distal renal tubular acidosis
(dRTA), which leads to metabolic acidosis, growth retardation,
hypercalciuria, and hypokalemia. Some patients with AE1
defects exhibit an inability to acidify the urine spontaneously
or following an acid load with no metabolic acidosis under
basal conditions, more commonly referred to as “incomplete
dRTA.”
Dominant and recessive forms of dRTA arise from AE1
mutations. Differing modes of inheritance are explained by
AE1 dimerization. Most AE1 mutations cause the protein to
misfold, resulting in intracellular retention, rather than traf-
ficking to the cell surface (100). In the case of dominant muta-
tions, the intracellular retention phenotype of the mutant is
dominant over the wild-type protein in heterodimers, whereas
in the case of recessive disease, the heterodimers traffic nor-
mally to the cell surface (100). Mislocalization of kAE1 to the
apical renal tubular membrane has also been identified as the
basis for dRTA in some patients (101,102). Recent reports
have confirmed the role of the C-terminal motif in trafficking
of AE1 to the plasma membrane (103).
dRTA incidence is especially high in malaria-endemic
parts of Southeast Asia: Papua New Guinea, Thailand, and
Malaysia. Interestingly, individuals with this nine amino acid
deletion in AE1 correspond to the N-terminus of the first trans-
membrane segment of the protein (amino acids 400–408). In
the heterozygous state, individuals develop ovalocytic red
blood cells, a benign condition called Southeast Asian ovalocy-
tosis (SAO; ref. 104). SAO individuals are heterozygous for the
trait and asymptomatic (except for the shape of their red
cells). SAO-AE1 is, however, nonfunctional as a Cl2/HCO32
exchanger (105). This benign condition arises from a tighten-
ing of the interaction between AE1 and the cytoskeleton, which
limits the ability of the Plasmodium falciparum parasite to
invade erythrocytes. Thus, individuals recessive for the SAO-
AE1 allele are resistant to malaria, leading to an evolutionarily
driven increase in the incidence of the allele. Until very
recently, SAO-AE1 homozygotes had not been observed, pre-
sumably as the genotype is seriously deleterious in utero. Con-
sistent with this notion was the recent first report of a child
homozygous for the SAO allele, who was prematurely born,
Alka and Casey
Role of AE2 in gastric parietal cell acid secretion.
FIG 6 Polarized gastric parietal cells are responsible for
secretion of HCl into the stomach lumen. To do so,
CO2 (a conjugate acid) diffuses from the blood into
the cell. Cytosolic carbonic anhydrase II (CAII) cata-
lyzes conversion of CO2 to H1 and HCO32. AE2-
mediated Cl2/HCO32 exchange concomitantly loads
the cell with acid (by base removal) and provides
Cl2. HCl is secreted into the stomach lumen through
concerted action of the gastric H1/K1-ATPase and a
Cl2 channel. The mechanism of acid secretion onto
the bone surface by bone-resorptive osteoclasts is
similar. SLC26A7 may also carry out Cl2/HCO32
exchange or function as a Cl2 channel in parallel to
AE2.
manifested severe anemia (treatable by transfusion), and
developed dRTA (106). dRTA is common among SAO individu-
als, as SAO-AE1 is not functional in anion exchange, and thus,
mutation of their second AE1 allele leaves them with greatly
compromised AE1 function.
Ae12/2 mice have fragile erythrocytes as a consequence of
disruption of the AE1 cytoskeletal connection (107,108). These
mice manifest serious anemia, cardiac hypertrophy, impaired
cardiac function, and dRTA, compromising their viability
(109,110).
AE2 (SLC4A2). AE2 is a widely expressed bicarbonate trans-
porter, whose Cl2/HCO32 exchange activity contributes to
homeostatic control of cell pH through acid loading, arising
from efflux of the base, HCO32, in exchange for influx of Cl2
(111). The membrane domain of AE2 shares 45% amino acid
identity with AE1; however, the AE2 N-terminal cytoplasmic
domain is divergent, with low sequence identity. Little is
known about its role, other than the contribution of amino
acids 310–347 to response of AE2 activity to changes of intra-
cellular and extracellular pH (112,113). There are five spice
variants of AE2 (a, b1, b2, c1, and c2) that arise as a result of
alternate splicing from three (a, b, and c) alternative promoter
sites. Major sites of AE2 expression include basolateral mem-
branes of choroid plexus, gastric parietal cells (Fig. 6), renal
collecting duct, and thick ascending limb (8,9,114). As
601

examples of AE2 function, in acid-secreting osteoclasts and
gastric parietal cells, AE2 basolateral Cl2/HCO32 exchange
loads the cell with Cl2 and prevents cytosolic alkalosis that
would otherwise arise on HCl secretion (Fig. 6). AE2 regulates
cytosolic pH and is thus steeply negatively regulated at acidic
cytosolic pH (112). AE2-mediated regulatory cell volume
increase (115) coordinates action of AE2 with a Na1/H1
exchanger, resulting in cytosolic NaCl loading, without pH
change, and osmotic fluid movement in the cell (116).
AE2 in Disease. Ae22/2 mice manifest growth retardation,
failure of tooth eruption, and gastric achlorhydria, as well as
mild gastric dysplasia, reflecting the critical role played by AE2
in acid loading in parietal cells (117). Loss of AE2 function in
these mice was lethal to the embryo and most died around the
time of weaning. AE2 and NBCe1, expressed on apical and
basolateral membrane of secretory ameloblasts, respectively
(118), have important roles in tooth development (119). The
key role of AE2 in acid secretion by bone-resorbing osteoclasts
(Fig. 6) means that loss of AE2 shifts bone balance toward
osteopetrosis or pathologically high bone density.
AE3 (SLC4A3). AE3 is comparable with AE2, in having a lon-
ger cytoplasmic N-terminal region than AE1 and pH sensitivity
of transport activity (113). Two alternate transcripts, bAE3
(brain AE3, also called full length AE3) and cAE3 (cardiac
AE3), are transcribed from alternative promoters (120,121).
AE3 is commonly referred to as the anion exchanger of excita-
ble tissue as its distribution is limited to brain (bAE3), retina
(bAE3 and cAE3), and cardiac tissues (cAE3).
AE3 in Disease. The AE3-A867D variant has been found in
some patients with idiopathic generalized epilepsy (122). Hetero-
logously expressed AE3-A867D had reduced functional activity
and processed normally to the cell surface. This AE3 variant
was thus suggested to promote epilepsy through alterations of
brain cell volume and pH homeostasis (123). Additionally, these
effects may be mediated through alterations of intracellular Cl2
concentrations, which would impact on the activity of GABA Cl2
channels, affecting neurotransmission. Consistent with a role in
epilepsy etiology, ae32/2 mice have a reduced threshold for
chemically induced seizure (124).
The retina is the most metabolically active tissue of the
human body, and AE3 has an important role in disposal of
metabolic waste HCO32 (pKa of the conversion of mitochon-
drial respiratory waste product CO2 to HCO32 1 H1 is near the
cytosolic pH, leading to the accumulation of significant level of
bicarbonate; Fig. 1). Indeed, ae32/2 mice can manifest visual
defects, arising from loss of AE3 from the inner retina (125).
Significant roles have been assigned to AE3 in the heart.
In heart failure, cardiac hypertrophy (increased cell size with-
out increased cell number) is a central feature. Pathological
concerted action of NHE1 and AE3 has been proposed to cause
cell loading with NaCl, leading to hypertrophic growth, in part
through an inhibitory effect on the plasma membrane Na1/
Ca21 exchanger, NCX1 (126). Consistent with this idea, cul-
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tured cardiomyocytes from ae32/2 mice are less responsive to
stimuli that induce hypertrophic growth than wild-type coun-
terparts (127). Further roles for AE3 in the heart came from
the study of ae32/2 mice crossed with tropomyosin-mutant
(TM180) mice, which suggested that loss of AE3 was not cardi-
oprotective against hypertrophic growth (128). The discrepant
effects of AE3 in cardiac hypertrophic growth may arise from
differences in genetic background of the mouse strains. Alter-
natively, the observation of protective effects of AE3 ablation
was made in cultured isolated cardiomyocytes (127), whereas
a protective role of AE3 expression was found in the studies of
intact hearts (128). Careful studies of ae32/2 mice suggested
that ae32/2 hearts responded to stress in a way similar to
hearts in failure, suggesting a key protective role of AE3 (129).
Indeed, one report found that an inhibitory anti-AE3 antibody
impaired recovery from alkaline load and in the response of
cardiac myocytes to endothelin stimulation (130).
Na1/Bicarbonate Cotransporters of the SLC4 Family
Na1/Bicarbonate Cotransporters (NBCs) cluster separately
from AE proteins phylogenetically (Fig. 2) and further divide
into electrogenic NBCs (NBCe1 and NBCe2) and electroneutral
NBCs (NBCn1, NBCn2, and NDCBE). The stoichiometry of
cotransport for both NBCe1 and NBCe2 is 1 Na1:2 HCO32/1
Na1:3 HCO32 influx or efflux depending on the tissue distribu-
tion, [Ca21], and phosphorylation status of the protein
(131–133). A number of transport models have been proposed
for NBCe1; however, no particular model can be assigned yet
(see ref. 134). The substrate that NBCs transport is under dis-
cussion. Some data suggest that NBCe1 can carry CO322
instead of 2 HCO32 (135,136). The physiological significance of
CO322 transport, however, remains unclear as CO322 levels
are much lower than HCO32 in blood. In blood at pH 7.4,
[HCO32] (pKa 6.4) is about 1,000-fold higher than [CO322] (pKa
10.3).
NBCe1 (SLC4A4). The first NBC identified was NBCe1 (e, for
electrogenic and number for order of identification), cloned
from salamander kidney (13), which led to the identification of
the human ortholog (14,137). NBCe1 has two domains: a cyto-
plasmic N-terminal domain and a 14-transmembrane segment
membrane domain, with a cytoplasmic C-terminal tail
(138,139). Interestingly, the cytoplasmic domain of NBCe1 has
residues critical for substrate bicarbonate entry into the mem-
brane domain (140), a feature that is not found in AE1 (141),
despite sequence conservation between the domains of the two
proteins. NBCe1 is dimeric with each subunit functioning inde-
pendently in transport activity (142). The site of highest
NBCe1-a expression is basolateral membrane of proximal
tubule, where it is plays a critical role in reabsorption of
HCO32 base into the blood (Fig. 7; ref. 143). NBCe1-b (N-termi-
nal 41 amino acids of NBCe1-a are replaced by 85 amino
acids) is widely expressed in basolateral membrane of pan-
creas (144,145), where it facilitates efflux of HCO32 into the
pancreatic duct (Fig. 3; ref. 146). NBCe1-c (46 amino acid of Ct
Bicarbonate Transporters in Health and Disease

Role of NBCe1 in renal proximal tubular NaHCO32
FIG 7 reabsorption. The human kidney proximal tubule
reabsorbs about 500 g/day of NaHCO32. Defects in
this process thus give rise to proximal tubular acido-
sis. In the renal tubule lumen, HCO32 is titrated with
H1 and converted to CO2 in a reaction catalyzed by
carbonic anhydrase IV (CAIV), anchored to the cell
surface by a glycosylphosphatidylinositol linkage.
CO2 diffuses across the apical cell surface into the
cytosol, where carbonic anhydrase II (CAII) catalyzes
conversion to HCO32 and H1. Apical NHE3 effluxes
the H1 in exchange for influx of Na1. At the basolat-
eral membrane, NBCe1 completes net uptake of Na1
and HCO32 into the blood.
of NBCe1-b is replaced by 61 amino acid) is expressed in
brain, where it contributes to pH regulation (147).
NBCe1 in Disease. NBCe1 has a central place in renal
Na1 and HCO32 reabsorption. NBCe1 at the basolateral mem-
brane of the proximal tubule is responsible for most renal Na1
reabsorption and acid secretion (reabsorption of the base,
HCO32 is equivalent to acid secretion) through efflux of HCO32
and Na1 from the tubule epithelial cell cytoplasm into the
blood (Fig. 7). Thus, NBCe1 defects cause proximal renal tubu-
lar acidosis (pRTA; refs. 148–150). R298S and R510H, the first
reported NBCe1 mutations, significantly reduce its transport
activity (55 and 57% of wild type, respectively; ref. 149).
Homozygous point mutations in NBCe1 are associated with
pRTA, glaucoma, and cataract. Mutations can give rise to dis-
ease through aberrant cellular targeting. For example, S427L
was mistargeted to the apical membrane, whereas R510H was
retained in intracellular membranes when expressed in
Madin-Darby Canine Kidney cells (151). Characterization of a
series of NBCe1 mutants revealed varied expression/activity in
different expression systems and a requirement of at least
50% or more reduction in activity to cause severe pRTA (152).
NBCe1 plays a role in normal cardiac function, suggesting
that heart disease may arise from NBCe1 dysfunction. Indeed,
NBCe1 activity contributes to the cardiac action potential
(153). Furthermore, one study using an inhibitory anti-NBCe1
Alka and Casey
antibody found that NBCe1 inhibition is cardioprotective in the
context of ischemic injury, likely by reducing accumulation of
Na1 following reperfusion (154).
Important roles of NBCe1 in eye function have been estab-
lished over the recent years. Endothelial cells in the posterior
cornea reabsorb fluid from the corneal stroma into the ante-
rior chamber of the eye. NBCe1, localized at the basolateral
surface, facing the stroma, is proposed to have a central role
in endothelial cell fluid movement (155). Loss of NBCe1 func-
tion causes vision disruption, ranging in severity from serious
progressive corneal opacification (156) to blindness (149).
Interestingly, studies of nbce12/2 mice reveal that the gene
is essential for normal tooth enamel development (157).
NBCe2 (SLC4A5). Among the seven NBCe2 transcripts (called
a–g), only two are functional, with a stoichiometry of 1:2/1:3
(Na1:HCO32; refs. 17 and 18) depending on the cell type (17,18).
It must be noted that some of these transcripts could be cloning
artifacts. Recently, NBCe2-g has been identified as the key
player in the efflux of HCO32 across the apical membrane of rat
choroid plexus (158). NBCe2 is widely expressed in tissues,
including liver, testes, brain, heart, lung, spleen, kidney, pla-
centa, and small intestine (19).
NBCe2 in Disease. Nbce22/2 mice manifest serious defi-
ciencies in regulation of cerebrospinal fluid (CSF) levels, con-
sistent with a major role of this transporter in choroid plexus
(159). A recent review of choroid plexus function in CSF pro-
duction features a prominent role for apical NBCe2 (160). Fur-
thermore, a neuronal role for NBCe2 is illustrated by the sensi-
tivity of these mice to develop seizures and their significant
retinal defects (159). A human genetic study revealed NBCe2 as
associated with salt-sensitive hypertension. Since all but one
single-nucleotide polymorphism (SNP) were in noncoding
regions, changes of gene expression may be responsible for the
effect (161,162). Supporting a role of NBCe2 in control of blood
pressure, careful analysis of slc4a52/2 mice revealed hyperten-
sion in the animals (163). Hypertension was proposed to arise
from loss of renal NBCe2 function, compensated by increased
expression and function of NBCn1, which is present in renal
collecting duct (164).
NBCn1 (SLC4A7). Originally called NBC3, the first identified
electroneutral NBC is now called NBCn1 and is widely distrib-
uted (heart, skeletal muscle, spleen, testis, brain, lung, liver,
and kidney). NCBn1 exists as splice variants (20,165–167).
NBCn1 expressed in muscle tissues (168) carries out cotrans-
port of 1 Na1 and 1 HCO32. In neurons, NBCn1 contributes to
maintenance of the resting pH preceding neuronal firing
(165). NBCn1, expressed basolaterally in rat inner medullary
collecting duct (169) and the thick ascending limb of Henle’s
loop (170), is upregulated on NH4Cl-induced chronic meta-
bolic acidosis (170).
NBCn1 in Disease. Nbcn12/2 mice have significant defects
in hearing and vision, reflecting a need for the H1-neutralizing
function of HCO32 moved by NBCn1 (171). Interestingly, the
603

combined auditory/visual defects in nbcn12/2 mice are similar
to Usher syndrome, the most common deafness/blindness syn-
drome in humans. Physical interactions between NBCn1 and
proteins defective in Usher syndrome, including the scaffold
protein Harmonin, led to the suggestion that NBCn1 is a key
component of a coordinated protein complex critical to control
of retinal and inner ear function (172).
A role of NBCn1 in control of vascular tone is also strongly
indicated. nbcn12/2 mice are mildly hypertensive, and these
mice show decreased responsiveness to the vasodilator, nitric
oxide (173). Studies of these mice indicate an important role of
NBCn1 in the regulation of vascular cytosolic pH.
NBCn2 (SLC4A10). NBCn2 (previously referred to as NCBE, for
Na1-dependent Cl2/Bicarbonate exchanger), primarily expressed
in brain, differs from NBCn1 functionally as it can carry out two
uncoupled transport activities: 1) Cl2 self-exchange in 1:1 ratio
(stimulated by HCO32), and 2) cotransport of 1 Na1 and 1 HCO32
(30). NBCn2 also facilitates Na1-dependent Cl2/HCO32 exchange
(30,174).
NBCn2 in Disease. Localization of NBCn2 to the basolateral
surface of the choroid plexus epithelium, combined with the
small brain ventricles in slc4a102/2 mice, strongly suggest a cen-
tral role of NBCn2 in CSF production (160,175). Protection
against serious epileptic seizures in the slc4a102/2 mice, expres-
sion of NBCn2 in hippocampal neurons, and significant decrease
in recovery from acid load also indicate a key role of NBCn2 in
homeostatic control of neuronal pH (175). Furthermore, blind-
ness arises in slc4a102/2 mice, which was suggested to occur
because of a failure to maintain appropriate cytosolic levels of
Cl2 and HCO32 (176).
NDCBE (SLC4A8). Na1-dependent chloride bicarbonate ex-
changer (NDCBE) is expressed mainly in central nervous
system. Unlike the other two electroneutral cotransporters,
NDCBE carries out electroneutral Na1-dependent Cl2/
HCO32 exchange with the stoichiometry of 1:2:1 (Na1/
HCO32/Cl2, inward/outward/inward; ref. 22).
NDCBE in Disease. High NDCBE expression is found in pre-
synaptic terminals in close association with synaptic vesicles,
most glutamatergic axon terminals, and terminals of
parvalbumin-positive c-aminobutyric acid (GABA)ergic cells
(177). Reduced acid extrusion in cultured hippocampal neurons
of slc4a82/2 mice along with increased seizure threshold in
vivo suggests that NDCBE regulates pH to control presynaptic
glutamate release (178). Characterization of slc4a82/2 mice led
to the conclusion that NDCBE activity forms part of the
thiazide-sensitive Na1 reabsorption pathway in the distal renal
tubule (179). Indeed, the emerging model is that in base-
secreting (type B) intercalated cells of the distal renal tubule,
apical SLC26A4 and NDCBE together accomplish net NaCl
reabsorption (179,180).
SLC4A9. Phylogenetically, SLC4A9 (also called AE4) clearly
clusters with Na1-coupled bicarbonate transporters (Fig. 2),
604
IUBMB LIFE
suggesting that the protein functions as a Na1-coupled trans-
porter. Yet, SLC4A9 cloned from the rabbit kidney (27) and rat
(181) displayed Cl2/HCO32 exchange activity when expressed
in HEK293 cells, leading to the name AE4. One preliminary
report found that AE4 is an electroneutral Na1-coupled bicar-
bonate cotransporter (28), which would be consistent with the
protein’s phylogeny. Localization of SLC4A9 to the basolateral
membrane of b-intercalated cells of the rabbit cortical collect-
ing duct, combined with reduced expression during acidosis,
are consistent with a role of the protein in efflux of Na1/
HCO32 from cytosol into blood (182).
SLC4A9 in Disease. Slc4a92/2 mice have no obvious phe-
notype, suggesting that the role of SLC4A9 is dispensable or
can be compensated by other genes (183).
Bicarbonate Transporters of the SLC26 Family
SLC26 family members function as Na1-independent electro-
neutral/electrogenic anion exchangers or as anion channels,
with broad substrate range, distinguishing them from the
SLC4 family of bicarbonate transporters. Important physiologi-
cal roles of this group of transporters are evident from its dis-
tinct distribution pattern and link to various disease states, as
described below. Of the 11 SLC26 genes, SLC26A10 is a pseu-
dogene, and only SLC26A3, 4, 6, 7, and 9 gene product trans-
port bicarbonate (Table 1).
Structure of SLC26 Proteins
Recently, the structure of SLC26 proteins has been reviewed
(184). SLC26 proteins have three distinct regions: a variable
cytoplasmic N-terminal region (51–106 amino acids), an inte-
gral membrane domain of 12 transmembrane segments, and a
cytoplasmic C-terminal STAS (sulfate transporter and anti-
Sigma factor antagonist) domain (185) related to those found
in some bacterial proteins. SLC26 proteins are dimeric (186),
as also seen for SLC4 proteins.
The integral membrane domain of SLC26 proteins has
been the subject of few publications. Studies of SLC26 glycosy-
lation site mutants led to detailed topology models for all
SLC26 proteins, each with 12 transmembrane segments and
cytosolic N- and C-termini (185). Homology modeling of
SLC26A6 on the basis of the E. coli ClC protein was able to
predict functionally important residues, providing limited sup-
port that SLC26 proteins share the ClC protein fold (45). None-
theless, there is an inconsistency as ClC proteins do not have
the 12-transmembrane segment structure that SLC26 proteins
have. A high-resolution structure for the membrane domain of
an SLC26 protein will be required to resolve the issue.
SLC26 STAS domains and adjacent sequences interact
with other transporters (e.g., citrate transporter NaDC-1),
cytoskeletal scaffolds, and with enzymes metabolizing trans-
ported anion substrates, thereby forming a transport metabo-
lon (for review, see ref. 187). The SLC26A6 STAS domain binds
CAII (188) and interacts with the regulatory R-domain of CFTR
(189). The STAS domain is of great significance as so many
disease-causing mutations localize to this region (187).
Bicarbonate Transporters in Health and Disease

Intestinal NaCl reabsorption. Intestinal NaCl uptake
FIG 8 drives osmotic fluid reabsorption, leading to diarrhea
when the process fails. In the cytosol of epithelial
cells lining the small intestine, carbonic anhydrase II
(CAII) converts CO2 into HCO32 and H1. These enzy-
matic products act as substrates for NHE3 and the
Cl2/HCO32 exchangers SLC26A3 and SLC26A6
(SLC26A3/6), respectively. NHE3-mediated H1 efflux
is coupled to Na1 uptake from the intestinal lumen.
SLC26A3 couples the HCO32 efflux to Cl2 uptake.
NaCl reabsorption into blood is completed by con-
certed action of the Na1/K1-ATPase and Cl2 and K1
channels. This is a simplified version of highly regu-
lated process, involving other proteins (190).
SLC26A3. SLC26A3 roles are predominantly at the apical
surface of pancreatic and intestinal epithelia (Fig. 8). Func-
tional studies of SLC26A3 yielded discrepant findings.
SLC26A3 is reported either as an electrogenic 2Cl2/HCO32
exchanger (33,191) or electroneutral Cl2/HCO32 exchanger
(187). Electroneutral transport would be consistent with a
role in the coupled electroneutral absorption of NaCl in small
intestine (187). In another set of experiments, involving
knockout mice, SLC26A3 also carries out DIDS-sensitive
HCO32/SO422 exchange (192). Species or cell-specific differ-
ences may explain the discrepancies in the electrogenicity of
transport (192). Recently, studies of slc26a32/2 mice suggest
that SLC26A3 mediates apical oxalate and chloride uptake in
small and large intestines (193). Phosphorylation of the
SLC26A3 STAS domain regulates its interaction with the reg-
ulatory domain of the CFTR (189). SLC26A3–CFTR interaction
activates CFTR Cl2 channel function (189), and the CFTR R-
domain activates SLC26A3 (194).
SLC26A3 in Disease. SLC26A3 was first identified in colon
as a candidate tumor suppressor gene, with a significantly
reduced expression in adenomas and adenocarcinomas. Thus,
it was referred to as downregulated in adenoma (DRA; ref.
195). One of the original names for SLC26A3 was CLD, because
30 mutations in the gene have been identified to cause autoso-
mal recessive Chloride Losing Diarrhea (34,35). Normally,
Alka and Casey
SLC26A3 and the NHE3 Na1/H1 exchanger facilitate concerted
colonic reabsorption of NaCl (Fig. 8). Loss of SLC26A3 compro-
mises the process and the associated osmotic water uptake;
failed fluid reabsorption leads to catastrophic diarrhea (35).
slc26a32/2 mice had significant neonatal mortality, whereas
survivors suffered high-chloride diarrhea, volume depletion,
and growth retardation (36). Furthermore, these mice exhibited
significant reduction in apical Cl2/base exchange with acidic
luminal content in colon with compensatory adaptive upregula-
tion of ion-absorbing transporters (e.g., NHE3, H1/K1-ATPase,
and sodium channel; ref. 36). Finally, mutations in the
SLC26A3 STAS domain cause misfolding and/or mistrafficking,
leading to the loss-of-function of the protein (196).
SLC26A4 (Pendrin). The SLC26A4 gene product, commonly
called Pendrin, is an electroneutral Cl2/HCO32 exchanger
(39) also able to transport Cl2, HCO32, I2, formate, thiocya-
nate, and NO32 (39,40). Pendrin is expressed in the apical
membrane of nonsensory epithelial cells of the outer sulcus
and spiral prominence of the cochlear duct, transitional cells
surrounding the vestibular neuroepithelia, and mitochondria-
rich cells of the endolymphatic sac (41). In addition, apical
expression of pendrin was found in salivary duct, thyroid fol-
licles (197), b-intercalated cells of cortical collecting duct
(Fig. 4; ref. 198), and airway epithelia (199).
SLC26A4 in Disease. SLC26A4 mutations cause autosomal-
recessive Pendred syndrome, a relatively common genetic disease
(1/10,000 approximate incidence), characterized by congenital
deafness and goiter (200–202). Most SLC26A4 mutants are
retained in intracellular membranes, with significant heterogene-
ity in cellular localization, degree of N-glycosylation, and sensitiv-
ity to therapeutic rescue (203).
In thyroid folliculocytes, SLC26A4 mediates CI2/I2 exchange,
responsible for iodide efflux to the follicle colloid. Thus, human
SLC26A4 mutations that impair ion translocation fail to secrete
iodide in thyroid follicles, resulting in goiter (202). Slc26a42/2
mice have no thyroid phenotype (204,205), suggesting that other
mechanisms contribute to iodine secretion in mice. Interestingly,
in some individuals, SLC26A4 mutations cause hearing loss,
without goiter, for unclear reasons (201,206).
Hearing loss in Pendred syndrome arises from loss of
SLC26A4 HCO32 secretory function into the endolymph of the
inner ear (201,207). The decreased pH of endolymph has been
proposed to impair hearing by inhibition of acid-sensitive Trpv5
and Trpv6 Ca21 uptake channels (207). Loss of pendrin function
in nonsensory epithelia of inner ear (cochlea, vestibular organs,
and endolymphatic sac) is associated with the enlargement of
the vestibular aqueduct (206,207). In mice with SLC26A4
expression targeted to endolymphatic sac but not in cochlea/
vestibular organs, normal hearing was restored without any
swelling of inner ear (208), indicating that endolymphatic
SLC26A4 function is sufficient to support normal hearing.
SLC26A4 has significant roles in the kidney. In base-
secreting (type B) intercalated cells of the distal nephron, api-
cal SLC26A4 secretes HCO32 into the renal tubule in exchange
605

for tubular Cl2, predominantly under conditions of metabolic
alkalosis, to regulate systemic pH (Fig. 4; ref. 198). Slc26a42/2
mice manifest acidic urine and downregulated epithelial cal-
cium channel and Na1/Ca21 exchanger (causing hypercalciuria)
in the kidney (209). As discussed earlier, recent studies indicate
that NDCBE and SLC26A4 accomplish net NaCl uptake in type
B cells, with potential significance for hypertension (179,180).
Kidney-targeted slc26a4 ablation leads to elevated HCO32 lev-
els in serum, consistent with failure of HCO32 secretion (210).
SLC26A6. SLC26A6 is expressed in pancreas, intestine, kidney,
heart, skeletal muscle, and placenta. SLC26A6 transports OH2,
SO422, formate, oxalate, and NO32 anions; however, there are
species and splice variant-specific differences in coupling stoichi-
ometry (46,47,211,212). Mouse SLC26A6 expressed in Xenopus
laevis oocytes carries out electrogenic Cl2/HCO32 exchange with
isoform specific stoichiometry (1:2; refs. 33 and 191). Human
SLC26A6 expressed in X. laevis oocytes, however, exhibited elec-
troneutral Cl2/HCO32 and Cl2/OH2 exchange with comparable
rates. In addition, human SLC26A6 had much slower chloride
and sulfate transport when compared with the mouse ortholog
(212). Cl2/HCO32 exchange activity of human SLC26A6 was
stimulated by cAMP and CFTR, but inhibited by the cystic fibro-
sis allele, F508del CFTR (212). Hence, the varying anion coupling
stoichiometry might depend on species and expression system.
SLC26A6 in Disease. Homozygous slc26a62/2 mice were
normal in terms of blood pressure and kidney function. Abol-
ished oxalate-stimulated NaCl absorption, however, decreased
apical Cl2/base exchange activity in the knockouts (213). Insol-
uble calcium oxalate is a key component of kidney stones. The
presence of calcium oxalate stones in kidneys of slc26a62/2
mice (214) suggests that variants of human SLC26A6 might
contribute to stone formation, although this is yet to be estab-
lished. Fructose ingestion induces hypertension, as a result of
increased NaCl absorption. Interestingly, the phenomenon is
absent in slc26a62/2 mice, leading to a hypothesis of fructose
stimulation of SLC26A6 (by means unknown), which increases
intestinal NaCl absorption, leading to hypertension (215). Ele-
gant experiments on slc26a62/2 mice demonstrated the role of
SLC26A6 acting in concert with NHE3 to achieve electroneutral
salt absorption in the small intestine (Fig. 8; ref. 216). Further-
more, a role of SLC26A6 in HCO32 efflux at the apical mem-
brane of pancreatic ducts has been identified (Fig. 3; ref. 217).
SLC26A7. SLC26A7 localizes to the basolateral membrane of
gastric parietal cells (218), type A intercalated cells of the
outer medullary collecting duct (219), proximal tubule, and
thick ascending limb of the loop of Henle (220). SLC26A7 facili-
tates electrogenic Cl2/HCO32 exchange (221) and has Cl2
channel function (222). Oxalate and sulfate are also trans-
ported, albeit more slowly than Cl2 (50).
SLC26A7 in Disease. No human diseases have yet been
definitively associated with SLC26A7; however, the critical
roles of the protein identified through mouse studies strongly
suggest diseases associated with the protein. SLC26A7 func-
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tions predominantly as Cl2/HCO32 exchanger in acid-secreting
cells of outer medullary collecting duct (colocalized with AE1;
Fig. 4). Thus, deletion of slc26a7 in mice decreased basolateral
HCO32 efflux, resulting in bicarbonate wasting which leads to
dRTA (51). Similarly, in stomach, SLC26A7 functions in acid-
secreting gastric parietal cells (colocalized with AE2; Fig. 6),
and thus, slc26a72/2 mice displayed decreased gastric acid
secretion possibly due to impaired HCO32 efflux or enhanced
Cl2 entry in parietal cells (51).
SLC26A9. SLC26A9 is expressed in brain, airway epithelial
cells (223), gastric parietal cells (53), and collecting duct of
kidney (224). SLC26A9 functions in three modes: 1) electro-
genic Cl2/HCO32 exchange, 2) chloride channel, and 3)
Na1:Cl2 or HCO32 cotransport (53,225–227).
SLC26A9 in Disease. slc26a92/2 mice have a significant
reduction of renal chloride excretion under high salt diet or
when subjected to water deprivation (224). This work sug-
gested an important role played by SLC26A9 in renal chloride/
fluid excretion and arterial pressure regulation, linking
impaired SLC26A9 directly to hypertension (224). Structural
abnormalities in gastric parietal cell acid secretory apparatus
in slc26a92/2 mice implicate SLC26A9 in normal parietal cell
function (226). Finally, in human bronchial epithelial cells,
SLC26A9 interacts with CFTR, where it acts as a Cl2 channel
under basal and cAMP/protein kinase A-stimulated conditions.
More specifically, the role of SLC26A9 in human bronchial
cells requires functional CFTR (227).
SLC26A11. SLC26A11 (also called KBAT [kidney brain anion
transporter]) was initially described as a SO422 transporter
(228). Guinea pig SLC26A11 expressed in X. laevis oocytes did
not transport HCO32, but did transport chloride, oxalate, and
sulfate (48). Cl2/HCO32 exchange activity has been reported
only for mouse SLC26A11 (229). Human SLC26A11 facilitates
sulfate transport (228), although its ability to transport HCO32
has not apparently been assessed. Taken together, it is uncon-
vincing that SLC26A11 can be regarded as a bicarbonate
transporter. Recently, it was suggested that SLC26A11 is a Cl2
channel, regulating acid translocation by H1-ATPase across
the plasma membrane and in intracellular compartments in
Purkinje cells (230).
Bicarbonate Transport Metabolon
CAs are zinc metalloenzymes (EC 4.2.1.1) catalyzing reversible
hydration of CO2 to HCO32 and H1 (1). The 10 active CAs differ
in catalytic kinetics and cell location. CAII, the most widely dis-
tributed cytosolic isoform, has an enormous catalytic rate of
about 106 s21. The extracellular membrane-associated iso-
forms are CAIV, CAIX, CAXII, and CAXIV. As CAs both produce
and consume the substrate of bicarbonate transporters,
HCO32, CA activity affects the rate of bicarbonate transport
(231).
Considerable evidence led to the idea of a “bicarbonate
transport metabolon” (71). A metabolon is a physical complex
of sequential enzymes in a metabolic pathway, where physical
Bicarbonate Transporters in Health and Disease
association reduces loss of material outside the pathway and
thus maximizes the coupled flux through the pathway (232). A
bicarbonate transport metabolon is a complex between a
bicarbonate transporter and the cytosolic/extracellular CAs
responsible for production and consumption of the transported
HCO32. Initial evidence of AE1/CAII interaction (233) was fol-
lowed by identification of a specific electrostatic interaction
between a basic patch in the N-terminal region of CAII and a
CA-binding motif (hydrophobic residue, followed by four amino
acids, at least two of which are acidic) in the cytosolic C-
terminus of AE1 (234–237). Later, CAII/AE1 interaction was
found to accelerate AE1 transport rate (71). Elegant evidence
in support of the bicarbonate transport metabolon model
found that heterodimers of AE1, with one subunit able to bind
CAII (but with a functionally inactivating mutation) and the
other subunit transport component (but with a mutation block-
ing CAII binding), was activated by CAII (238). Further, ration-
ale for the need for the transport-accelerating effect of CAII
association with erythrocyte AE1 is the slow rate of H1 move-
ment at the membrane surface, combined with extremely fast
capillary transit time of red cells during which Cl2/HCO32
exchange must be completed (239).
CA enzymes with their catalytic domain anchored to the
extracellular surface also activate bicarbonate transporters.
CAIV interacts with the fourth extracellular loop of AE1, form-
ing the extracellular component of the bicarbonate transport
metabolon (93,240). AE3 physically interacts with CAII and
extracellular CAXIV (125). AE2 is functionally activated on
binding extracellularly linked CAIX (241).
The mechanism for enhanced transport activity on associ-
ation with CAII has been proposed as follows: 1) localization of
CAII, which promotes substrate channeling to and from the
anion translocation pathway; 2) interaction of CAII with AE1
increases CAII catalytic rate (242); and 3) H1/monocarboxylate
transporters (MCTs) are activated by association with CAs
(243–245). One insight from these studies was the suggestion
that transport is increased by an enhanced rate of H1 move-
ment near the transporter by the polybasic sequence on the
CAII N-terminus. This was explained as an “H1-antenna” that
rapidly guides H1 in the vicinity of an associated transport
protein (246). Intriguingly, catalytic activity of CAs is not
required to increase MCT function, suggesting that the
enhancement arises from the H1-antenna effect.
NBC interaction with CAs has also been reported. A C-
terminal cytoplasmic region of NBCn1 interacts with cytoplas-
mic CAII with a Kd of 101 nM (247). NBCe1 when expressed
with different isoforms of CA (I, II, and II) in X. laevis oocytes
showed increased transport activity (248). Two acidic motifs in
human NBCe1, L958DDV and D986NDD, interact physically
with CAII (249). Extracellular CAIV and NBCe1 localize in kid-
ney and pancreatic duct cells, where NBCe1 plays a central
role in bicarbonate absorption into the blood and HCO32
influx, respectively (250). The fourth extracellular loop
of NBCe1 interacts with CAIV to increase its transport
activity (250).
Alka and Casey
Among SLC26 proteins, there is diversity in bicarbonate
transport metabolon phenomena. SLC26A3 does not have a
consensus CAII-binding site and correspondingly does not bind
CAII; however, SLC26A3 transport activity is activated by CAII
catalysis (251). In contrast, the SLC26A6 STAS domain binds
CAII in a phosphorylation-regulated manner, consistent with
regulation of transport function by modulation of CAII binding
(188).
The bicarbonate transport metabolon model has, however,
come under critical scrutiny. In one report, in vitro assays
were unable to find evidence for specific interaction of AE1
peptides with CAII (252). Evidence has also been presented
that NBCe1 is not activated by CAII interaction (253). Recently,
the CAII–AE1 interaction came under renewed investigation
(254). In particular, the article reported the following factors:
1) CAII and AE1 were unable to be coimmunoprecipitated, a
finding previously attributed to low affinity of the interaction.
2) Lack for FRET between N-terminally fluorescent protein
tagged AE1 and C-terminally tagged CAII, suggesting a lack of
interaction. The possibility of steric interference by the fluores-
cent proteins is a concern, as CAII and the fluorescent protein
are of similar size and the CAII crystal structure reveals the N-
terminal AE1-binding site and the C-terminus to be close to
each other. 3) Lack of effect of CAII on the rate of HCO32
membrane permeation. This result could be explained by the
presence of endogenous CAII in the cell line providing suffi-
cient catalytic activity to saturate AE1. The assays in this case
also measured AE1 transport activity at baseline rate of
HCO32 permeation. The effects of CAII under high rates of
AE1 transport activity, as done earlier (71), were thus not
assessed.
Taken together, the catalytic activity of CA enzymes and
the transport activity of bicarbonate transporters are clearly
linked processes. Whether bicarbonate transporters are also
activated by localization of CA to their surface remains
controversial.
Cancer and Bicarbonate Transport
The metabolic shift in solid tumors toward hypoxia, glycolytic
metabolism, and acidic extracellular pH is well established.
Investigators are working toward targeting these changes in
design of cancer therapeutics (255). Indeed, the extracellular
anchored CA, CAIX, is proving a promising drug target (256).
Recently, attention has also turned toward the contribution of
bicarbonate transporters to cancer cell biology, and the associ-
ation of bicarbonate transporter gene mutations in cancers
has been quantified (257). Bicarbonate transporters through
their regulation of the pH of cancer cell cytosol and extracellu-
lar space have potential to alter rates of cancer cell
proliferation.
AE2 expression is increased in colon tumors, relative to
neighboring noncancerous tissue. Moreover, suppression of
AE2 expression in a colon cancer cell line decreased cell pro-
liferation, together suggesting a role of AE2 in promoting
607

growth of colon cancer cells (258). The observation is not
entirely explicable as prevailing ion gradients greatly favor
HCO32 efflux by AE2, thus promoting cellular acidification. It
is not clear how enhanced cytosolic acidification could
enhance cell growth.
Among NBCs, NBCe1 has been reported as either underex-
pressed or overexpressed depending on cancer cell type, pre-
cluding a simple explanation of the role of the NBCe1 in can-
cer (257). Genome-wide association has linked a SNP in the
SLC4A7 gene, encoding NBCn1, to risk of breast cancer. As
the SNP is in the 30 -untranslated region of the gene, risk is
likely associated with aberrant regulation of gene expression,
rather than altered function of NBCn1 (257). More directly,
NBCn1 expression is elevated in breast cancer tissue (259),
most simply explained by a role of NBCn1 in alkalinizing can-
cer cell cytosol.
SLC26 proteins, most prominently SLC26A3, have pro-
posed roles in cancer (257). SLC26A3 was originally called
DRA, reflecting its decreased expression in colorectal adenoma
and adenocarcinomas (195). Supporting a growth-suppressive
role of SLC26A3, overexpression of the protein in cancer cell
lines reduced cell growth (260), and slc26a32/2 mice manifest
increased colonic cell growth (36). Finally, SLC26A4 expres-
sion is elevated in some thyroid tumors, leading to the sugges-
tion that it may provide a selective advantage to these cells by
increasing cytosolic pH (257).
Conclusions
A large group of researchers over the recent decades have
uncovered the repertoire of proteins responsible for bicarbon-
ate transport in mammals. The roles of these proteins in nor-
mal physiology and in disease are increasingly becoming clear.
The importance of these proteins in fundamental processes,
including gastric acid secretion and bone resorption, suggests
that specific bicarbonate transport modulators would be excel-
lent drugs. Identification of modulators of individual bicarbon-
ate transporters will be an important topic for future research.
In the near future, we can expect high-resolution structures
for representatives of the SLC4 and SLC26 families. These
structures will provide molecular mechanisms for diseases
caused by mutations of bicarbonate transporters in many
cases. Only in the recent years has a role of bicarbonate trans-
porters in cancer become a focus of study. In the future, we
anticipate that this will become a major research focus, which
may lead to cancer therapies.
Acknowledgements
Research in the Casey Laboratory is supported by the Cana-
dian Institutes of Health Research. The authors thank Dr.
Emmanuelle Cordat and Katie Badior for helpful comments on
the manuscript. The authors are grateful to the investigators
in this research area and apologize for any work uncited in
the article.
608
IUBMB LIFE
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