The family of mammalian bicarbonate transport proteins are involved in a wide-range of physiological processes. The importance of bicarbonate transport follows from the biochemistry of HCO3− itself. Bicarbonate is the waste product of mitochondrial respiration. HCO3− undergoes pH-dependent conversion into CO2 and in doing so converts from a membrane impermeant anion into a gas that can diffuse across membranes. The CO2–HCO3− equilibrium forms the most important pH buffering system of our bodies. Bicarbonate transport proteins facilitate the movement of membrane-impermeant HCO3− across membranes to accelerate disposal of waste CO2, control cellular and whole-body pH, and to regulate fluid movement and acid/base secretion. Defects of bicarbonate transport proteins manifest in diseases of most organ systems. Fourteen gene products facilitate mammalian bicarbonate transport, whose physiology and pathophysiology is discussed in the present review.
- bicarbonate transport
- Cl−/HCO3− anion exchanger (AE)
- Na+/HCO3− co-transporter (NBC)
- pH regulation
- solute carrier (SLC)
- volume regulation
Bicarbonate (HCO3−) has a central position in mammalian physiology. Mitochondrial respiratory oxidation produces CO2 as its oxidative waste product; CO2 is in equilibrium with HCO3−+H+. Since the pKa of 6.4 for this conversion is near physiological cytosolic pH (approx 7.2), at equilibrium both HCO3− and CO2 are present at significant levels (Figure 1). The ability of HCO3− to undergo pH-dependent conversions is central to its physiological role. The primary buffer of our bodies is the CO2/HCO3− buffer system. CO2 is a conjugate acid, which freely diffuses across membranes, thus representing a membrane-permeant acid equivalent (Figure 1). Conversely, since HCO3− is a base, the cytosolic pH rises as the molecule enters the cell and falls as it exits the cell. Since HCO3− is charged it cannot move across membranes without facilitation by bicarbonate transport proteins, which are integral membrane proteins. Mammalian bicarbonate transporters, the focus of the present review, have physiological roles reflecting the chemistry of HCO3−: disposal of waste CO2/HCO3−, regulation of cellular and whole-body pH, acid/base secretion and fluid secretion.
Multicellular organisms face a challenge in moving membrane-impermeant bicarbonate from inside the cell where it is produced to the environment for disposal. The nematode, Caenorhabditis elegans, illustrates the requirement for bicarbonate transport proteins in multicellular organisms. C. elegans' genome contains twelve paralogues of mammalian bicarbonate transporters ; bicarbonate transport is thus significant in the physiology of even small multicellular organisms. Bicarbonate transport activity has, however, only been established for one of the C. elegans gene products .
In mammals 14 genes encode proteins identified as bicarbonate transporters (Table 1). The physiological roles of these bicarbonate transporters and the diseases that arise from aberrant bicarbonate transporters are the focus of the present review. This review presents examples of the major roles of bicarbonate transporters, but is incomplete in light of the breadth of physiological roles of bicarbonate transport.
MAMMALIAN BICARBONATE TRANSPORTERS
Phylogeny of bicarbonate transporters
In mammals there are 14 genes which encode proteins with bicarbonate transport activity (Table 1). Table 1 also summarizes the coupling stoichiometry or electrogenicity of the transport activity of these gene products. In some cases some ambiguity remains in aspects of transporter function, especially physiological substrates and coupling stoichiometry. As in any research field there is some disagreement in the literature and Table 1 is based on our parsing of the literature.
Bicarbonate transporter genes were identified over a period of more than 20 years [2,3], often with multiple independent identifications. These discoveries were superimposed with physiological identification of bicarbonate transport activities, without knowledge of the responsible gene. Together, this led to a confusing nomenclature for bicarbonate transporters. The Human Genome Organization has applied a systematic nomenclature to human genes, where membrane proteins facilitating movement of soluble substrates are classed as solute carriers or ‘SLC’ . Using this categorization bicarbonate transporters are in the SLC4A and SLC26A families, which have distinct evolutionary origins, and are sufficiently dissimilar to be considered to be separate families of proteins. Amino acid sequences of human bicarbonate transporter genes were analysed on the basis of sequence similarity to infer genetic relationships (phylogeny) (Figure 2). Within SLC4A, there is a clear phylogenetic division into two groups (subfamilies), functionally corresponding to electroneutral Cl−/HCO3− exchangers and Na+-coupled HCO3− co-transporters (Figure 2).
The nomenclature for bicarbonate transporters is still inconsistent, but is settling with time. In the present review, we have used a nomenclature that is becoming accepted and which distinguishes the genes in a meaningful way (Table 1 and Figure 2). In some cases the systematic nomenclature is used, in particular where no abbreviated name exists to describe the transport function of the gene. In one case (pendrin) the name comes from the disease (Pendred syndrome) caused by defects in the transporter , which has become the common name for the gene.
Electroneutral Cl−/HCO3− exchangers
Electroneutral anion exchangers were the first bicarbonate transporters to undergo detailed physiological and molecular analysis. Initial studies were driven by the high abundance (50% of integral membrane protein) of the Cl−/HCO3− exchanger in the RBC (red blood cell) membrane, coupled with the simplicity of isolation of RBC and their plasma membrane. The RBC Cl−/HCO3− exchanger was the first bicarbonate transporter cloned and sequenced. The gene was called AE1 (anion exchanger 1) , recognizing that there were likely to be other genes in the family. Hydropathy analysis of the 911 amino acid mouse AE1 amino acid sequence predicted a protein with 12–14 transmembrane segments [2,6]. Subsequent extensive protein chemical studies refined the topology model, and identified more structural and functional details (Figure 3) (summarized in ). The two domain structure of the protein is composed of an N-terminal 43 kDa cytoplasmic domain which binds the RBC cytoskeleton and glycolytic enzymes, and a 55 kDa membrane domain able to facilitate transport alone . The AE1 gene is expressed as two alternate transcripts, eAE1 (erythrocyte AE1), expressed only in RBCs, and kAE1 (kidney AE1), which is expressed only in the basolateral membrane of acid-secreting type A-intercalated cells of the renal collecting duct.
cDNA libraries screened with probes based on the mouse AE1 sequence revealed two additional members of the AE1 family, AE2 and AE3 [9,10]. AE1–AE3 genes revealed a shared architecture. AE2 and AE3 differ from AE1 in having larger N-terminal cytoplasmic domains (approx. 70 kDa) and in the third extracellular loop of AE2 and AE3, which is larger than the second and serves as the site of N-linked glycosylation  (Figure 3). Considerable divergence emerges in the amino acid sequences of cytoplasmic domains, but the high conservation in their membrane domains is consistent with a conserved transport function.
Like AE1, AE3 has a restricted pattern of expression: heart, brain and retina, leading to the generalization that AE3 is the AE of excitable tissues, without evidence for polarized expression. Alternative promoter usage generates two different AE3 gene products, AE3fl (AE3 full length) and AE3c (AE3 cardiac) . In contrast, AE2 is the ‘housekeeping’ isoform of the Cl−/HCO3− exchanger, with near ubiquitous expression. Although AE2 regulates cytosolic pH through efflux of cytosolic HCO3− for extracellular Cl−, it has distinct roles in acid secretion in many physiological settings. Alternative promoter usage generates three N-terminal variants of AE2 protein, with tissue-specific expression . AE2 and the kAE1 variant are both basolaterally localized in epithelia, whereas AE3 is not associated with epithelial tissues.
The AE family of Cl−/HCO3− exchangers share many common features, but differ in some ways. All function by an electroneutral mechanism, exchanging Cl− for HCO3− across the plasma membrane, driven by the respective gradients of the transport substrates. There is no mechanistic barrier to reversible transport, as illustrated by AE1–AE3-mediated HCO3− influx and efflux driven by reversing the transmembrane Cl− gradient when expressed in transfected cells . Under physiological conditions only RBC AE1 functions reversibly; RBC AE1 effluxes HCO3− in peripheral capillaries and influxes HCO3− at the lungs, driven by reversal of the transmembrane HCO3− gradient in the two settings. AE2 and AE3 facilitate cytosol acidification through HCO3− efflux in exchange for Cl−, driven by prevailing Cl− and HCO3− gradients.
Na+-coupled HCO3− co-transporters
Na+-coupled HCO3− transporters cluster phylogenetically (Figure 2), indicating descent from a common ancestor and suggesting a common function. Common among these transporters is HCO3− transport coupled to the movement of Na+. Since mammalian cells have a large inward-directed Na+ gradient this transport mechanism provides a driving force for HCO3− accumulation into the cell. That said, the electrogenic Na+/HCO3− co-transporter, NBCe1, has a key role in renal Na+ and HCO3− reabsorption, acting to move its substrates from the cytosol to the blood in the basolateral surface of proximal tubule cells, driven by the negative membrane potential and net negative charge movement associated with NBCe1 activity.
The HCO3− transport mechanism and the direction of HCO3− transport (influx versus efflux) are complicated by the diversity of coupling stoichiometries, leading to differences in electrogenicity of HCO3− transport (Table 1). Indeed, NBCs can be electroneutral (NBCn1) or electrogenic (NBCe1, NBCe2). Moreover, the coupling stoichiometry of NBCe1 is either 2 or 3:1 (HCO3−/Na+), depending on the site of NBCe1 expression and phosphorylation status of the protein [15–17]. Preliminary evidence suggested that NBCe1 can transport CO32− . The physiological significance of CO32− transport is, however, uncertain. Blood pH is approx. 7.4 and the pKa for CO32− and HCO3− protonation are 10.3 and 6.4 respectively (Figure 1). Together this implies that the [CO32−] in blood is approx. 100-fold lower than [HCO3−]. Therefore either NBCe1 is highly selective for CO32− over HCO3−, or the amount of CO32− transport is small in comparison with HCO3− transport.
SLC4A9 (where SLC is solute carrier) is the controversial member of the Na+-coupled HCO3− transporter family. The protein was originally called AE4 and reported to facilitate Cl−/HCO3− exchange . No other laboratory has reported Cl−/HCO3− exchange activity for the gene; our laboratory was also unable to detect Cl−/HCO3− exchange activity in SLC4A9 transfected HEK (human embryonic kidney)-293 cells (D. Sterling and J. Casey, unpublished work). Combined with the strong phylogenetic clustering of AE4 with bona fide Na+-coupled HCO3− co-transporters (Figure 2), consensus has emerged that AE4 is a Na+-coupled HCO3− co-transporter, not a Cl−/HCO3− exchanger [20,21].
Finally, two different human genes have been reported to encode Na+-dependent Cl−/HCO3− exchangers: NDCBE (SLC4A8) and NCBE (SLC4A10) (Table 1). Human NDCBE cDNA, expressed in Xenopus laevis oocytes, acted as an electroneutral Na+-dependent Cl−/HCO3− exchanger, with the stoichiometry 1:2:1 (Na+/HCO3−/Cl−) . Using the same expression system, human NCBE had the same functional activity as NDCBE . Extracellular Cl− stimulated Na+ uptake and Cl− efflux, consistent with NCBE-mediated Cl−/Cl− exchange. A recent re-examination found that NCBE was able to facilitate Cl−/Cl− self-exchange and Cl−-independent electroneutral Na+/HCO3− co-transport . On the basis of this study, NCBE joins NBCn1 as an electroneutral NBC; NCBE has thus been proposed to be re-named NBCn2 .
Whether SLC4A11 belongs among HCO3− transporters is uncertain. When the SLC4A11 gene was identified it was called BTR1 (bicarbonate transporter 1) , on the basis of sequence similarity to AE1–AE3, although SLC4A11 does not cluster strongly with the AEs phylogenetically (Figure 2). Yet no bicarbonate transport activity has been reported for SLC4A11 protein, so there is no evidence that SLC4A11 is a HCO3− transporter. Fascinatingly, SLC4A11 has sequence similarity to the plant borate transporter, bor1 . In line with this, human SLC4A11 acted as a sodium/borate co-transporter when expressed in transfected mammalian cells . The significance of the finding is clouded, however, as there is no established biochemical role for borate in mammals. Resolving the issue of SLC4A11 transport activity does have poignancy, since the gene has recently been identified as defective in three corneal dystrophies [28–30].
Cl−/HCO3− exchangers of the SLC26 family
The third group of bicarbonate transporters is the SLC26 family, which are of special interest because of their links to human genetic diseases [31,32] (Table 2). Although the SLC4 bicarbonate transport proteins are sufficiently similar to be regarded as arising from a common ancestor, amino acid sequence identity between SLC4 and SLC26 proteins is too low to regard them as evolving from a common gene. Initially identified as sulfate transport proteins [33,34], some SLC26 proteins function as Cl−/HCO3− exchangers (Table 1). Although there are ten members of the SLC26 family , only SLC26A3, 4, 6, 7 and 9 are established as bicarbonate transporters.
The transport activity of bicarbonate transporters of the SLC26 family remains somewhat controversial. There is evidence for electrogenic Cl−/HCO3− exchange by both SLC26A3 and SLC26A6 [35–37], but the transport stoichiometry of these two important bicarbonate transporters has been contentious. SLC26A3 has been reported to facilitate 1:≤2 HCO3−/Cl− exchange [36,37] or electroneutral exchange , whereas SLC26A6 stoichiometry is either 2:1 (HCO3−/Cl−) [36,37] or electroneutral . Yet, careful physiological studies of mice with knockouts of Slc26 genes have suggested that both Slc26a3 and Slc26a6 are electroneutral [38,40]. At present the evidence suggests that Slc26a3 and Slc26a6 are electroneutral, but species or cell-specific differences may yet prove to explain the observed electrogenic behaviour of these bicarbonate transporters. SLC26A4 facilitates electroneutral Cl−/HCO3− exchange . Transport activity of SLC26A7 is still under debate. Cl−/HCO3− exchange activity of SLC26A7 has been reported independently [3,42–44] and Cl−-coupled anion-exchange activity has also been observed for SLC26A7 . In contrast, SLC26A7 expressed in X. laevis oocytes acted as a pH-regulated Cl− channel [45,46], suggesting an electrogenic mechanism for the transporter. SLC26A9 has received little attention to date and controversy remains as to whether the protein is a Cl−/HCO3− exchanger, or a Cl− channel, possibly modulated by HCO3− [47–49]. The preponderance of evidence led us to call these proteins ‘electrogenic Cl−/HCO3− exchangers', differentiating them from SLC4 proteins (Figure 2).
A unifying feature of the SLC26 proteins is the presence of the STAS (sulfate transporter and anti-σ factor antagonist) domain in the C-terminal cytoplasmic region. The STAS domain, initially identified in sulfate transporters and anti-σ antagonists, has an unclear role in the function of SLC26 transporters, but is involved in protein–protein interactions. The STAS domain SLC26A6 forms a CA (carbonic anhydrase) II-binding site  and interacts with the regulatory R-domain of the CFTR (cystic fibrosis transmembrane conductance regulator) Cl− channel . The significance of the domain is underscored by the number of disease-causing mutations that localize to the STAS domain and by subtle mutations in the plant orthologue, SULTR1.2, that abrogate its sulfate transport [31,32,52].
OTHER BICARBONATE/CARBON DIOXIDE TRANSLOCATION MECHANISMS
Although the present review focuses on bicarbonate transport proteins, a full rendering requires consideration of other mechanisms that contribute to transmembrane bicarbonate flux.
CO2 membrane permeation
Since CO2 is a conjugate acid, transmembrane CO2 movement is equivalent to acid movement, such that diffusion of CO2 into a cell acidifies the cell, whereas efflux of CO2 alkalinizes it. Diffusion of CO2 is physiologically significant; approx. 25% of acid efflux during recovery of an ischaemic heart is attributable to CO2 diffusional efflux, in comparison with approx. 50% of pH recovery by influx of alkaline HCO3−, mediated by bicarbonate transport proteins .
Although CO2 diffusion across lipid bilayers is well-established, whether CO2 permeation occurs through transport proteins is far more controversial. Excellent research has provided evidence for and against permeation of CO2 through AQP (aquaporin) water channels [54,55]. AQP1 has been proposed to be part of a macro-complex involving the entire bicarbonate transport apparatus of RBCs: AQP1, the AE1 and CAII . CO2/HCO3− exchange is thus facilitated through co-localization of the metabolic/transport machinery. Indeed, RBCs from humans lacking AQP1 revealed that the protein is responsible for 60% of transmembrane CO2 flux . The significance of AQP1 to CO2 permeation may be limited to RBCs as strong evidence also suggests that AQP1 is not involved in CO2 permeation in lung and kidney , two other tissues where CO2 membrane flux is important. Finally, modelling studies suggest that CO2 permeation through AQP1 may only be physiologically significant in membranes with low gas permeability or in cells with high expression levels of AQP1 . The importance of facilitation of CO2/HCO3− permeation is underscored by the identification of the RBC Rhesus complex protein, RhAG, as a CO2 channel .
Members of the SLC39A family of metal transporters (ZIP8 and ZIP14) mediate HCO3− transport [60,61]. The two transport proteins function as Zn and Mn transporters in testis and kidney, but can also carry Cd, implicating them in Cd toxicity. ZIP8 is proposed to use the transmembrane HCO3− gradient to drive metal uptake . Although ZIP8 and ZIP14 formally have bicarbonate transporter activity the total bicarbonate flux through these pathways probably contributes little to HCO3− homoeostasis.
HCO3− conductive anion channels
Some Cl− channels are HCO3− permeable and contribute to physiological HCO3− flux. This is not surprising since Cl− and HCO3− are both small anions and some bicarbonate transport proteins (i.e. Cl−/HCO3− exchangers) readily accept Cl− and HCO3− as substrates. The CF (cystic fibrosis) gene product, the Cl− channel called CFTR, is 25% as permeable to HCO3− as Cl−, leading to the suggestion that CFTR provides a large fraction of epithelial HCO3− secretion, in pancreas , uterine endometrial cells  and in duodenal HCO3− secretion .
Control of pH is crucial to neuronal function, given the high metabolic rates of acid production and sensitivity of electrical flow to changes of pH. Neurotransmitter receptors for GABA (γ-aminobutyric acid) and glycine both have inherent Cl− channel activity, opened upon agonist binding. The ability of HCO3− to permeate both GABA and glycine receptors has been recognized for over 20 years . The negative neuronal membrane potential implies that GABA receptors and glycine receptors mediate HCO3− efflux. In the confined space of the synaptic cleft this permeation may result in pH changes and increased HCO3− levels with implications for bicarbonate transporters.
Recently human bestrophin Cl− channels (Best) were recognized as HCO3− conductive . Indeed, bestrophin isoforms hBest1, 2, 3 and 4 (where hBest is human Best) are all HCO3− conductive, with conductance ratios for HCO3−/Cl− ranging from 0.6–1.1, indicating HCO3− conductance nearly as high, or higher than, Cl− conductance. The widespread expression of Best channels and the high conductance of HCO3− may force reconsideration of HCO3− permeation mechanisms in some tissues.
PHYSIOLOGY AND PATHOPHYSIOLOGY OF BICARBONATE TRANSPORT
Common features are found in the physiological roles of bicarbonate transporters and the mechanisms of diseases associated with loss of their function. In general, bicarbonate transporters serve to: (i) facilitate efflux of the respiratory CO2/HCO3− load, (ii) regulate cell pH, (iii) excrete HCO3− and (iv) contribute to cell volume regulation. Mutations of bicarbonate transporters impair transport function by either: (i) impairing transport function or (ii) impairing cellular targeting, either by mis-targeting to the wrong epithelial surface or retention in the ER (endoplasmic reticulum).
The oligomeric state of bicarbonate transporters affects the inheritance pattern of genetic diseases of these proteins. The basic structural unit of both SLC26 and SLC4 bicarbonate transporters is a dimer [67–69]. Most disease-causing mutations of bicarbonate transporters have an ER-retained phenotype; thus heterodimers of WT (wild-type) and mutant proteins may be ER-retained, causing a dominant pattern of inheritance.
RBCs facilitate the transport of oxygen and carbon dioxide in the blood. The RBC plasma membrane contains 106 copies of the Cl−/HCO3− exchanger, AE1, which is essential for optimization of respiration and stabilization of the plasma membrane. Since the AE1 gene encodes two different gene products RBC AE1 (commonly called Band 3) and kAE1 (expressed in the distal renal tubule), defects in the AE1 gene can cause RBC and renal defects. Yet, RBC and renal defects are rarely found in the same patient. In addition, some mutations can be dominantly inherited for RBC defects, but recessively inherited for renal pathologies. This phenotype is probably attributable to cell-specific differences.
AE1 maximizes the capacity of the blood to carry HCO3−/CO2 and has thus been proposed to be rate-limiting to whole-body respiration . Metabolic waste CO2 diffuses from cells into the plasma and from there into RBCs, where CAII facilitates conversion into HCO3−. Intracellular accumulation is prevented by AE1-mediated efflux of HCO3− in exchange for Cl−. At the lungs the low CO2 level drives a reversal of the process; AE1 transports HCO3− into the RBC, where CAII converts it into CO2, which is exhaled from the lungs. This CO2/HCO3− disposal cycle is enhanced by the physical interaction of the ‘DADD’ motif located in the cytosolic C-terminal domain of AE1 with CAII [71,72]. CAII catalyses the formation of bicarbonate and protons from CO2 and water. CAII forms a complex with AE1 termed a bicarbonate transport metabolon . This metabolon significantly increases the rate of bicarbonate transport , which is important since RBCs pass through a capillary in only 0.3–1.0 s.
AE1 is central to the RBC cytoskeleton, contributing to the distensibility observed as they pass through fine capillaries. AE1 is present as dimers and tetramers in the plasma membrane of RBCs . In the tetrameric form, AE1 interacts via its N-terminal cytosolic domain with ankyrin , which binds the cytoskeletal proteins α- and β-spectrin (Figure 4) . The cytosolic N-terminal domain of AE1 also interacts with glycolytic enzymes, haemoglobin, protein 4.1 and 4.2 , and their physical association with AE1 is regulated by phosphorylation of the cytosolic N-terminus of the exchanger .
Diseases of RBC AE1
Pathologies of RBCs linked to AE1 cause: (i) defects in mechanical properties, including spherocytosis and elliptocytosis, and (ii) passive flux of monovalent cations across the membrane (stomatocytoses) . Expansion of the inner membrane leads to membrane invagination and cup-shaped RBCs, called stomatocytosis. Stomatocytosis is caused by an increased ‘passive leak’, characterized by temperature-dependent excessive Na+ and K+ leakage from these cells, with no reduction of deformability . Hereditary stomatocytosis is caused by point mutations within the transmembrane segments of AE1 that confer cation channel activity upon AE1 . Elliptocytosis occurs in a remarkably high frequency (up to 39%) in Malayan and Melanesian aboriginal populations [79,80].
HS (hereditary spherocytosis) is the most common of the inherited RBC membrane disorders. Spherocytes are osmotically fragile in part because their spheroid shape reduces the reserve of volume in hypotonic medium. HS results from defective connections between the cytoskeleton and the plasma membrane (Figure 4). The common features of HS are hyperhaemolysis and anaemia, icterus and splenomegaly. RBCs from HS patients are spheroid with a reduced cell surface . HS mutations are located within cytosolic and transmembrane domains of AE1 and are found in the heterozygous or homozygous state. HS mutations are generally dominantly inherited and patients are usually heterozygotes. Three cases, however, report homozygous HS patients.
Band 3 Coimbra (V488M mutation) was found in the homozygous state in an anaemic newborn who had a massive hepatosplenomegaly [76,82,83]. Both eAE1 and kAE1 were missing. The RBCs of the child exhibited a wide variety of abnormal morphologies. Intensive care and blood transfusions allowed survival of the patient, who developed dRTA [distal RTA (renal tubular acidosis)] (see kidney-associated pathologies) .
Band 3 Neapolis was found in a homozygous patient with a deletion of the 11 N-terminal residues of Band 3 . Truncation of AE1 resulted from a single base substitution in the donor splice site. The RBCs of the patient displayed approx. 12% of normal AE1, due to a reduced stability of the aberrant mRNA and a complete loss of binding of aldolase and other glycolytic enzymes to AE1. The amount of other AE1-interacting proteins [GPA (glycophorin A), protein 4.2] was also reduced. This patient, however, did not develop renal-associated disease (dRTA) since these 11 N-terminal residues are absent from the kidney isoform, kAE1 (whose sequence begins at amino acid 66 of RBC AE1).
Band 3 Courcouronnes (S667F mutation) was found in a patient with HS and incomplete dRTA . RBCs from this patient displayed a 65% reduction of AE1 content in the RBCs of the patient, concomitant with a sharp reduction of GPA, protein 4.2 and the Rhesus complex (RhAG, Rh polypeptides and CD47), three proteins involved in a Band 3 macro-complex associated proteins at the plasma membrane of RBCs. In the Xenopus oocyte expression system, co-expression of Band 3 Courcouronnes with Band 3 WT or GPA did not improve the trafficking of the mutant AE1 protein, but slightly enhanced functional activity. In polarized epithelial MDCK (Madin–Darby canine kidney) cells, Band 3 Courcouronnes was predominantly retained in the ER.
Two lines of AE1-null mice have been characterized. The first was engineered to express kAE1, but not RBC AE1 . The second expressed neither the RBC AE1 nor kAE1 . Both lines of mice displayed similar symptoms: at birth, the Ae1−/− mice are extremely pale and 85% do not survive more than two weeks after birth. Survivors reach adulthood but are severely anaemic, and are smaller than their littermates. Adult Ae1−/− mice display splenomegaly and increased levels of immature RBCs (reticulocytes), indicating a compensatory increase in erythropoiesis. Ae1−/− RBCs are dehydrated microcytic cells, showing spherocytosis and poikilocytosis (RBC with abnormal shape), with rod-like membrane extensions. These cells lack both AE1 protein and protein 4.2, indicating that the two proteins interact as part of the RBC cytoskeleton.
Southeast Asian ovalocytosis
SAO (Southeast Asian ovalocytosis) is a haematological condition caused by a nine amino-acid deletion in the AE1 Cl−/HCO3− exchanger at the boundary between the N-terminal cytosolic domain and the first transmembrane segment [87,88]. Individuals heterozygous for the SAO trait have ovalocytic, more-rigid RBCs than normal , but are otherwise usually asymptomatic . SAO is found in regions where malaria is endemic and it may confer resistance to malaria . Although SAO AE1 can form heterodimers with normal AE1 , it does not fold into a transport-competent conformation [93,94]. Individuals with SAO are uniformly heterozygous for the trait; homozygosity is lethal in utero . When expressed in HEK-293 and MDCK cells, SAO AE1 was retained in the ER but when co-expressed with the normal AE1, SAO AE1 was partially targeted to the plasma membrane in both non-polarized and polarized MDCK cells . As SAO is exclusively found in the heterozygous state, rescue of trafficking by the WT isoform in kidney epithelial cells explains the lack of renal symptoms in these individuals.
Blood group antigens
Some mutations in the AE1 gene form antigens of the Diego blood group. Twenty-one mutations belonging to the Diego blood group are recognized by the International Society of Blood Transfusion . Prominent among Diego antigens is the Wright (Wra) blood group antigen (E658K) , and the Dia antigen (P854L) (Figure 3). Antibodies produced against Diego antigens cause transfusion reactions and haemolytic disease of the newborn.
RENAL HCO3− TRANSPORT
The kidney is the most active location of HCO3− transport in the body. Kidney avidly reabsorbs HCO3− to prevent its urinary secretion and the acidosis that results from secretion of this base from our bodies. RTA is a syndrome characterized by hyperchloraemic metabolic acidosis secondary to abnormal renal acidification. Proximal and distal parts of the kidney participate to the efficient reabsorption of HCO3− into the blood and H+ excretion into the urine. Defective bicarbonate transport in these locations leads to pRTA (proximal RTA) or dRTA.
Most filtered bicarbonate (80%) is reabsorbed by the proximal tubule in the kidney. Bicarbonate is freely filtered from the glomerulus into the tubular lumen. Filtered bicarbonate combines with protons in a reaction catalysed by the extracellular GPI (glycosylphosphatidylinositol) membrane-anchored CAIV . The CO2 produced diffuses into the proximal cells where it is reconverted into bicarbonate and water via cytoplasmic CAII (Figure 5). Proximal tubular cells secrete H+ by apical H+-ATPase and NHE3 (Na+/H+ exchanger 3). Basolateral NBCe1 facilitates movement of HCO3− into the blood . Mutations in the NBCe1 gene thus cause autosomal dominant pRTA [100–107]. Studies of NBCe1 expressed in Xenopus oocytes and in mammalian expression systems indicated that these mutations impair transport function  or cause mis-trafficking of the mutated NBCe1, retention in the ER [105,108] or apical mis-targeting .
SLC26A6 at the tubular surface of distal portions of the proximal tubule facilitates Cl−/HCO3− exchange, but no human mutations have been found to present with dRTA. The renal phenotype of Slc26a6-null mice reflects a defect in oxalate handling, reflecting the capacity of SLC26A6 to transport both oxalate, in addition to Cl− and HCO3− .
AE1 is expressed at the basolateral membrane of type-A (acid-secreting) intercalated cells, where it is involved in the fine-tuning of bicarbonate reabsorption and urine acidification (Figure 5). AE1 mutations cause dRTA, characterized by difficulties to thrive, renal stones, hypokalaemia, hyperchloraemia, metabolic acidosis and defective urine acidification. Alkaline therapy (potassium citrate or NaHCO3) indirectly corrects bone disorders and reduces the risk of nephrocalcinosis and renal stones. Complete dRTA appears when patients display spontaneous metabolic acidosis, whereas incomplete dRTA is generally diagnosed after an acid challenge, and the individual does not have any other symptoms . Mice lacking both erythroid and renal AE1 isoforms recapitulates renal features of dRTA patients, including hyperchloraemic metabolic acidosis, low net acid excretion, hypercalciuria, hypocitraturia and nephrocalcinosis with mild renal insufficiency .
Twelve mutations in the human SLC4A1 gene result in dRTA , yet only two out of 12 mutations result in both haematological defects and dRTA [82–84]. Band 3 Coimbra-induced complete dRTA was found in homozygous and heterozygous patients. Defective trafficking of dominant dRTA mutants induces mis-targeting of the WT subunit as well, due to heterodimerization [113,114]. Thus dRTA ensues as approx. 25% of basolateral WT AE1 homodimers (according to Mendelian rules) is not sufficient for required bicarbonate reabsorption. In contrast, defective trafficking of the recessive dRTA mutant AE1 is corrected by heterodimerization with the WT kAE1, thus resulting in the lack of dRTA symptoms [113,115].
In the dRTA mutant, R901X, truncating the last 11 amino acids of AE1 causes mis-targeting of the protein to the apical, instead of basolateral, membrane [116,117]. Apical R901X thus transports bicarbonate to the urine instead of to the blood, which explains the metabolic acidosis and elevated urine pH of these patients. Finally, the Cl−/HCO3− exchanger, SLC26A7, localizes with kAE1 to the basolateral surface of intercalated cells , suggesting that it may provide some HCO3− reabsorption capacity even when AE1 is disrupted.
SLC26A4, also called pendrin, facilitates exchange of Cl− for bicarbonate, sulfate and formate. Pendrin localizes to the apical surface of type B (base-secreting) and non-A, non-B type intercalated cells of the distal convoluted tubule, connecting tubule and cortical collecting duct, where pendrin transports HCO3− into the renal tubule [119,120]. Pendrin-knockout mice do not display any obvious kidney or thyroid dysfunction. An aldosterone analogue, however, induced up-regulation of pendrin expression in WT mice; associated metabolic alkalosis was more severe in pendrin-knockout mice than in WT mice. WT mice also gained more weight and developed more hypertension compared with pendrin-null mice . Pendrin-null mice display more acidic urinary pH and decreased pCO2, with reduced numbers of non-A type intercalated cells, without change of type-A intercalated cells . Together these results support a role of pendrin as the apical Cl−/HCO3− exchanger of base-secreting (type B) intercalated cells of the distal tubule. On a severe NaCl restriction, pendrin-null mice are hypotensive , indicating a role of pendrin in fluid balance.
Bicarbonate transporters do not have a specific function in thyroid gland activity. SLC26A4, however, is a dual function Cl−/HCO3− exchanger and Cl−/I− exchanger. SLC26A4 Cl−/I− exchange activity is required for I− efflux at the apical surface of thyrocytes ; abnormalities of SLC26A4 (as in Pendred syndrome) thus induce goiter.
Bicarbonate transport has two roles in intestinal function: neutralization of stomach acid entering the intestine and water reabsorption. Neutralization of acid by bicarbonate is primarily accomplished by the high bicarbonate concentration (125 mM) present in pancreatic fluid secreted into the intestine. In addition, CFTR-mediated bicarbonate efflux serves as the primary acid and cAMP/cGMP-stimulated bicarbonate secretion pathway in duodenum, and its activity is coupled to that of SLC26A6 . SLC26 family Cl−/HCO3− exchangers present in the apical membrane of intestine facilitate basal HCO3− secretion into the intestinal lumen and Cl− reabsorption, working in concert with CFTR . Water reabsorption by Cl−/HCO3− exchangers is achieved by co-operative action with the apical NHE3 Na+/H+ exchanger. Working together, NHE3 and Cl−/HCO3− exchangers reabsorb NaCl, with water following osmotically. The consequences of failed intestinal Cl−/HCO3− exchange manifest in the disease congenital chloride diarrhoea, resulting from mutations in the SLC26A3 Cl−/HCO3− exchanger [38,126]. In contrast with these HCO3− secretory mechanisms, at the apical surface of the proximal colon NBCe1 plays a role in HCO3− reabsorption . SLC26A6 is the predominant Cl−/HCO3− exchanger in the upper villus membrane of the duodenum . Two reports suggested a new mechanism for colonic HCO3− secretion; the high abundance of colonic SCFAs (short-chain fatty acids) drives SCFA/HCO3− exchange [128,129]. Whether this activity is directly facilitated by a transport protein or occurs through coupled action of multiple transporters (e.g. SCFA/H+ co-transport and Cl−/HCO3− exchange) awaits further study.
ACID SECRETION: STOMACH/BONES/TEETH
Sustained acid secretion challenges pH-regulatory mechanisms since failure to load cells with acid sufficient to compensate for that secreted will dangerously alkalinize cells. Gastric parietal cells, osteoclasts and ameloblasts involved in tooth formation exemplify the function of HCO3− transporters in cellular pH regulation.
Gastric parietal cells
Acid-secreting gastric parietal cells and bone-reabsorbing osteoclasts achieve different physiological roles, but have central features in common. In both cell types, HCl secretion at the apical surface requires Cl−/HCO3− exchange activity at the basolateral surface of the cell (Figure 6). Osteoclasts and parietal cells express basolateral AE2 to load cells with Cl− for secretion at the apical pole and concomitantly to remove HCO3− for acid loading. In both cell types AE2 is present in quiescent cells and acid secretion is initiated by insertion of vesicles containing the acid-secreting apparatus (H+-ATPase isoform and Cl− channel) to create a secretory canaliculus. Ae2−/− mice are achlorhydric (do not secrete HCl) , suggesting that AE2 is responsible for parietal cell HCl loading. Although SLC26A7 is a Cl−/HCO3− exchanger found at the basolateral surface of parietal cells, its significance is unclear, but SLC26A7, has been proposed to act as a Cl−-loading mechanism . Finally, SLC26A9 has been proposed to protect gastric mucosa by secretion of HCO3− on to the surface epithelium .
Enamel, synthesized by ameloblasts, forms in two stages: the secretory amelogenesis and the maturation stage. This second stage requires the formation of hydroxyapatite crystals in the enamel compartment, which generates 4–14 H+ per hydroxyapatite, which need to be neutralized to a physiological pH for the mineralization process to occur . Cellular mechanisms by which ameloblasts maintain intracellular and extracellular pH are just beginning to be defined. Ae2−/− mice are edentulous (toothless) , with impaired enamel maturation . Also, patients with NBCe1 mutations and mice lacking Nbce1 have chalky incisors prone to enamel fracture [101,104,127], suggesting roles for AE2 and NBCe1 in enamel formation. In mice, AE2 and NBCe1 are at the apical and basolateral membranes of secretory ameloblasts respectively . In contrast, AE2 localizes to the basolateral membrane during the maturation of ameloblasts in incisors where it may be functionally linked to cytosolic CAII and apical v-H+-ATPase, similarly to gastric parietal cells or osteoclasts .
BICARBONATE TRANSPORT AND THE BRAIN
The importance of pH regulation to brain function (reviewed in ) and heavy CO2/HCO3− load that comes with high energy production make HCO3− transport critical. Electrical activity of the brain induces rapid, localized changes of pH and HCO3− transporters assist in the control of local pH. Many HCO3− transporters have been identified in brain (Table 1), yet a specific role beyond pH regulation and disposal of HCO3− load remains elusive.
Bicarbonate transport acts in the pathophysiology of epilepsy, although the exact role is uncertain. Loss of AE3 Cl−/HCO3− exchanger function increases propensity for seizure: a point mutation in AE3 causes idiopathic generalized epilepsy . Ae3−/− mice also display a reduced threshold for chemically induced seizures . Conversely mice with a disruption of their Slc4a10 (NBCn2) gene have an increased threshold for chemically triggered seizures . The Slc4a10−/− mice had disrupted control of hippocampal neural networks, probably resulting from the loss of the pH-regulatory function of Slc4a10 . The use of CA inhibitors (acetazolamide, topimirate) as anti-epileptic drugs  suggests that altering HCO3− metabolism and probably HCO3− transport is effective in the control of epilepsy. This may follow from effects on the HCO3− conductive GABA receptor, which has a major role in both neural excitation and epilepsy; altering neural HCO3− levels by bicarbonate transporters could thus affect epilepsy through GABA receptor modulation.
CSF (CEREBROSPINAL FLUID) REGULATION
CSF has a composition similar to plasma . Although the [HCO3−] in plasma and CSF is nearly identical, plasma and CSF HCO3− levels are independently regulated, with CSF composition tightly controlled by the choroid plexus . The importance of HCO3− transport to CSF homoeostasis is illustrated by the expression of AE2, NBCe2, NBCn1, SLC26A7 and NBCn2 in choroid plexus [139,140]. Praetorius  has presented a comprehensive model for the role of these HCO3− transporters working at the apical (CSF-facing: NBCe2) and basolateral (blood facing: AE2, NBCn2, NBCn1) surfaces of the choroid plexus and their role in regulation of CSF Na+ and HCO3− levels and fluid homoeostasis. Reduced CSF production in Nbcn2-null mice suggests a significant role of the protein at the basolateral surface of choroid plexus .
Bicarbonate transport is significant to eye physiology. The retina is the most metabolically active tissue of our bodies, thus generating a large CO2/HCO3− load from respiratory oxidation that must be moved from the retinal site of production, to the bloodstream. Since blood vessels may occlude the light path of the eye, HCO3− may need to pass through avascular cell layers to reach the bloodstream, underscoring the need for effective bicarbonate transport pathways. An unidentified Cl−/HCO3− exchanger has been proposed to play a role in ciliary body aqueous humour production . Within the eye HCO3− transport has been implicated to have the greatest impact in the corneal endothelium and the retina.
The cornea, the outer surface of the eye, functions to provide physical protection to the eye and forms the initial path of light. The cornea has a multilayer structure, with an outer epithelial layer, a stromal layer and an inner endothelial cell layer, which in spite of its name is better considered as a reabsorptive epithelium. HCO3− transporters have significance to inner endothelium. The corneal stroma has a high concentration of dissolved solutes, in the form of proteoglycans, which presents a large osmotic driving force for water accumulation. Countering this the endothelial cell layer is active in ion transport, which drives fluid reabsorption from the stroma into the aqueous humour . The surface facing the aqueous humour is the apical cell surface. The role of HCO3− transporters in endothelial cell fluid reabsorption was initially suggested by the observation that the process required HCO3− and reabsorption was inhibited by CA inhibitors (reviewed in ). As carefully analysed by Bonanno , a basolateral NBC, probably NBCe1 , is required for endothelial cell function . Lending support to this, patients with mutations of NBCe1 manifest glaucoma, cataracts and band keratopathy [101–104,106,107]. Immunolocalization, RT (reverse transcription)–PCR analysis and functional assays indicate that basolateral AE2 plays a role in endothelial cell fluid reabsorption mechanisms . Since no eye phenotype has been reported for Ae2−/− mice [130,144], the function of AE2 in the endothelium may be compensated by another gene. Electrophysiology and ion-replacement experiments indicate an anion flux across the apical membrane that could be carried by HCO3− . If so, the HCO3− flux is conductive, possibly carried by CFTR; since individuals with CF have clear corneas and only a 4% increase in corneal thickness , the contribution of CFTR to endothelial fluid reabsorption is modest.
Corneal disorders are associated with many mutations of SLC4A11, which cause CHED2 (congenital hereditary endothelial dystrophy type 2), Harboyan syndrome and some cases of FECD (Fuch's endothelial corneal dystrophy) [28–30]. In CHED2 and FECD the endothelial cell layer is disturbed, causing loss of endothelial cells and consequent imbalance of stromal fluid levels. Descemet's membrane, a connective tissue layer secreted by the endothelial cells to create an adhesive layer between the stroma and endothelial layer, is also disrupted in CHED2. The disruptions manifest as an accumulation of collagenous material in Descemet's membrane that appears as bumps called ‘gutatta’ in microscopic biopsies . These gutatta combined with the stromal fluid accumulation impair vision significantly and interfere with quality of life for affected individuals. SLC4A11 diseases are inherited in a heterogenous manner; patients with Harboyan disease and CHED2 inherit SLC4A11 defects in a recessive manner [28,29,146], whereas FECD is dominant. Recessive mutations lead to an earlier onset, and more severe form, of the disease, owing to the absence of WT SLC4A11. SLC4A11, expressed in cornea, is responsible for these corneal diseases, and has a phylogenetic relationship with bicarbonate transporters (Figure 2), but the function of SLC4A11 remains uncertain, having been reported as a borate transporter . Whether SLC4A11 causes corneal diseases as a result of defective HCO3− transport remains unclear.
The retina is the most metabolically active tissue of the body with an associated heavy load of waste HCO3− . The exact mechanism of HCO3− transport through multiple cellular layers to reach the vasculature is not clear. The AE3 Cl−/HCO3− exchanger, however, is expressed in two differing splicing forms (AE3fl and AE3c), which are distinctly localized in either Müller glial cells or horizontal cells , suggesting unique functions for each. Significant visual impairment in Ae3−/− mice was revealed in their altered ERGs (electroretinograms) and structural changes of the retina, consistent with those found in human hereditary vitreoretinal degeneration disorders . Similarly, NBCn1 localizes to the retinal outer plexiform layer where it has an uncompensated role in pH regulation since Nbcn1−/− mice are blind . The phenotype of these mice could be explained by a failure to regulate pH in photoreceptor cells, which express Nbcn1. Loss of the alkalinizing effects of HCO3− influx mediated by NBCn1 would cause photoreceptor cell acidification and thus reduced driving force for the plasma membrane Ca2+/H+ exchanger that normally controls cytosolic Ca2+ levels in these cells .
The role of bicarbonate transport in hearing is illustrated by the range of diseases associated with mutations of HCO3− transporters. Prominent among these is Pendred syndrome, resulting from defects in the pendrin protein (SLC26A4). Pendrin is a Cl−/HCO3− exchange protein localized to the vestibular transitional cells and endolymphatic sac cells of the inner ear , which may be critical in regulating composition of the endolymph of the inner ear. Consistent with this explanation, pendrin-knockout mice display alterations in inner ear fluid resorption, resulting in excessive endolymph volume during a critical stage of inner ear development that may explain the congenital basis of Pendred syndrome . A more specific explanation of the link between loss of pendrin HCO3− transport function came with analysis of endolymph composition, which revealed that pendrin-knockout mice have acidic endolymph fluid, which impairs TRPV (vanilloid transient receptor potential) 5/6 Ca2+ channels, resulting in failed Ca2+ resorption and elevation of endolymph Ca2+ levels [153,154]. Elevated endolymph Ca2+ levels were proposed to impair sensory transduction required for hearing and to promote degeneration of sensory hair cells .
Knockout mice for the electroneutral Na+/HCO3− co-transporter, NBCn1, have a phenotype that mimics Pendred syndrome. NBCn1 localizes to the inner and outer hair cells of the organ of Corti . Nbcn1−/− mice manifest deafness associated with degeneration of sensory receptors, reminiscent of Pendred syndrome; however, no human disease has yet been linked to NBCn1.
A third potential bicarbonate transporter gene has been implicated in hearing defects. Harboyan syndrome, marked by corneal endothelial dystrophy and sensorineural hearing loss , was identified as caused by recessively inherited mutations in the SLC4A11 gene. cDNA microarray analysis of sub-regions of the ear revealed Slc4a11 transcripts in the mouse cochlea at the level of the lateral wall, which includes the stria vascularis . Again, defective endolymph ion homoeostasis could underlay this hearing deficiency. Harboyan syndrome remains enigmatic; as discussed above, SLC4A11 is clearly similar to bicarbonate transporters, but may have a different role. Furthermore, why the unique set of Harboyan mutations causes hearing loss, whereas another constellation of SLC4A11 mutations manifest as corneal defects without hearing loss, is unknown. Together Pendred and Harboyan syndromes and Nbcn1−/− mice suggest that control of endolymph bicarbonate homoeostasis is critical to hearing.
The importance of HCO3− transporters in cardiac function is explained by the high level of metabolic activity of the heart and the sensitivity of electrical activity to changes of pH. Indeed, HCO3− transport is estimated to be responsible for approx. 50% of pH regulation in heart . Contractile activity of cardiac muscle produces an acid load, largely in the form of CO2. Continued contraction requires disposal of waste CO2 and acid. During ischaemia, cardiac pH may drop as low as pH 6.2 , resulting from a failure to remove metabolic acid and anaerobic metabolism. Acid conditions directly impact cardiac function through: (i) closure of gap junctions that connect cardiac cells together to allow concerted contraction, this extreme sensitivity to low pH  may limit the coupling of healthy normo-pH cells to unhealthy acid cells; and (ii) alteration of ion-channel gating. Ion channels establish the membrane potential and signal contraction, thus arrhythmias result when acid pH alters channel gating.
Heart expresses an impressive range of bicarbonate transport proteins (Table 1). Given the prevailing Cl− and HCO3− concentration gradients Cl−/HCO3− exchangers in the heart represent an acidifying mechanism that also maintains the [Cl−] above electrochemical equilibrium . Analysis of Cl−/HCO3− exchanger mRNA abundance in mouse heart by real-time RT–PCR revealed that several Cl−/HCO3− exchangers were expressed, but Slc26a6 was the most abundant . Slc26a6 and Ae3 were not randomly distributed across the plasma membrane, but were concentrated at the T-tubule and in the intracellular membrane of the sarcoplasmic reticulum . This distinct pattern of localization suggests a role of Cl−/HCO3− exchange in pH regulation local to the Ca2+ handling apparatus of the myocyte.
A role of Cl−/HCO3− exchangers in the development of cardiac hypertrophy has been proposed, following on solid results indicating a role of the NHE1 Na+/H+ exchanger in the hypertrophic cascade [162,163]. NHE1 activity loads cardiomyocytes with Na+; this reduces the driving force for Ca2+ efflux by the plasma membrane Na+/Ca2+ exchanger and cytosolic Ca2+ elevation ensues. Under bicarbonate-free experimental conditions the activation of NHE1 causes a large rise in pHi (intracellular pH). Physiologically, however, there is abundant bicarbonate, meaning that the effects of bicarbonate-dependent pH regulatory processes are superimposed upon NHE1 activity. Widely ignored is the fact that when NHE1 is activated under physiological conditions (i.e. in the presence of bicarbonate) no concomitant rise of pHi is observed [164–166]. Why? NHE1 alkalinizes the cell but auto-inhibits at alkaline pH . Hyperactivation of NHE1 must be sustained by a balancing acid load. Interestingly, under hypertrophic stimulation cardiomyocytes do not show an increase in steady-state pHi [162,166], yet the increase of [Na+]cytosolic verifies that NHE1 is hyperactivated [168,169]. How is NHE1 hyperactivated without alkalinizing the cell? A parallel acidifying pathway, such as Cl−/HCO3− exchange, must be activated to balance the activity of NHE1 . AE3fl is the only Cl−/HCO3− exchanger whose activity is activated by PKC (protein kinase C) , the kinase that integrates hypertrophic pathways. Conversely, PKC inhibits SLC26A6 . A role of HCO3− transport in the hypertrophic cascade was further supported by the ability of CA inhibitors to prevent and revert hypertrophy in cardiomyocytes .
Na+-coupled HCO3− transporters
Na+-coupled HCO3− transporters have important roles in cardiac myocytes, in particular because of their electrical activity. Indeed, electrogenic NBC activity (NBCe1, NBCe2) shortens the cardiac action potential duration . NBCe1 expression levels were elevated in cardiomyopathic human hearts, whereas NBCn1 expression was unaltered in comparison with healthy controls. Furthermore, inhibition of NBCe1 activity with an antibody protected rat hearts from ischaemic injury .
The importance of HCO3− transport in CF pathology has begun to be recognized . In the pancreatic duct, CFTR is essential for secretion of fluid, rich in 140 mM HCO3−, in which enzymes are carried . Individuals with CF have a decrease in pancreatic fluid volume and a large decrease in [HCO3−] in the secretions. Pancreatic secretory defects in people with CF cause duct blockage, and fibrotic pancreatic destruction . Approx. 90% of individuals with CF have exocrine pancreatic insufficiency [62,176–178]. Correction of Cl− transport defects alone is not enough to ameliorate the symptoms of CF . Indeed, enhancing HCO3− transport by epithelial cells, even in the absence of CFTR, or increasing the HCO3− content on the apical surface of affected tissues should be considered as additional therapies to reduce the symptoms of CF. Defective bicarbonate secretion by CFTR manifests in intestine (see the section on the gastrointestinal tract above), pancreatic gland (see below) and lungs.
Pancreatic duct and airway are affected by loss of bicarbonate transport in CF. Pancreatic ducts express two Cl−/HCO3− exchangers of the SLC26 family: SLC26A6 and SLC26A3. The proximal pancreatic duct absorbs most of the Cl−, whereas the distal portion concentrates bicarbonate to 140 mM while absorbing the residual Cl−. The finding that luminal, but not basolateral, Cl−/HCO3− exchangers of the SLC26 family account for the regulation of this secretory/absorptive process supports such a model. In addition, since HCO3− is the main pH buffer, lack of HCO3− secretion disrupts the pH of fluids in luminal compartments, affecting the response of tissues to pathogens. This may be of special significance in the lungs where an acidic airway surface liquid from failed HCO3− secretion  may make the lungs of people with CF more prone to bacterial infection. Further significance of HCO3− transporters in CF came with the realization that CFTR functionally and physically interacts with NBCn1 (through the NBCn1 C-terminus) and with SLC26A6 (via the STAS domain) [36,51,181,182]. Finally, SLC26A9 localizes to the apical surface of bronchiolar and alveolar epithelia , which may prove to be important in the HCO3− secretory defects observed in CF-affected airways.
Salivary gland and pancreas
From the perspective of bicarbonate transport, pancreas and salivary gland have much in common: both are secretory tissues, secreting high levels of HCO3 . The mechanism responsible for accumulation of such a high concentration of HCO3− is a debated topic. There is, however, emerging consensus about the transporters involved, as shown (Figure 7).
Spermatogenesis occurs in a succession of stages that require cAMP-dependent processes triggered by a bicarbonate-sensitive AC (adenylate cyclase), recently identified as the sAC (soluble AC) . The bicarbonate transporter involved in the entrance of bicarbonate into developing spermatozoa and in epididymal epithelium has been identified as AE2. Homozygous Ae2−/− animals suffer high perinatal mortality . Homozygous Ae2−/− female mice are fertile, as are Ae2+/− male and female mice. Male Ae2−/− mice are, however, infertile, with a 40–60% reduction of testes size and weight, disruption of spermiogenesis and abnormal epididymal epithelia . However, no mutations of the AE2 gene have yet been linked to human infertility.
Sperm capacitation is the activation of fertilization capacity that occurs as sperm pass through the female reproductive tract. These changes include elevation of pHi and hyperpolarization of the sperm plasma membrane, all events dependent on extracellular HCO3− [186–188]. HCO3− ions activate a soluble cytosolic HCO3−-sensitive adenylate cyclase that leads to capacitation . CFTR, which mediates HCO3− flux, is expressed in sperm and is needed for sperm capacitation and motility. Two CFTR inhibitors reduced fertilizing capacity of sperm . Male Cftr+/− mice have reduced fertility. Infertile men have a 2-fold higher rate of heterozygosity for a CFTR mutation than the general population . In most cases, however, the fertility deficit results from congenital bilateral absence of vas deferens, a condition whose aetiology is unclear, but HCO3− transport could be involved.
Reduced fertility has been observed in women with CF. Although originally thought to result from abnormally thick mucus in the CF female reproductive tract that could form a physical barrier for sperm passage, the current view is that apical endometrial bicarbonate secretion has a dramatic role in sperm capacitation [192,193]. In the absence of extracellular chloride, mouse endometrial epithelial cells secrete bicarbonate through the apical membrane, when treated with the adrenergic agonist and CFTR activator, forskolin. Also, bicarbonate secretion is inhibited by the CFTR inhibitor, glibenclamide, suggesting that CFTR, rather than an anion exchanger, is involved. Sperm co-cultured with murine endometrial cells expressing low CFTR levels has reduced sperm capacitation and motility. In addition, sperm conditioned with medium collected from endometrial cells lacking CFTR results in reduced in vitro fertilization . This suggests that CFTR either mediates or regulates bicarbonate secretion into the female reproductive tract; CFTR defects may sufficiently reduce bicarbonate secretion to impair sperm capacitation and fertility.
Development of oocytes and embryos
Oocytes grow within ovarian follicles surrounded by granulosa cells. During development murine oocytes lack Cl−/HCO3− or Na+/H+ exchange activity. In fact, they do express a Cl−/HCO3− exchanger, whose identity is unknown, that remains quiescent due to the inhibitory action of the MAPK (mitogen-activated protein kinase) . Cl−/HCO3− exchange is thus regulated by the developmental status of the oocyte and is cell-cycle-dependent during meiosis. During early phases of oocyte growth, neutral pH in these cells is facilitated by granulosa cells via gap junctions; inhibiting gap junctions resulted in decreased Cl−/HCO3− exchange from follicle-enclosed oocytes, and in impaired recovery from induced alkalosis . Once fertilized, the early embryos maintain pH homoeostasis by releasing inhibition of the Cl−/HCO3− exchanger by MAPK .
Human bicarbonate transport proteins have now probably all been identified (Table 1). Their roles in pH regulation and bicarbonate metabolism are known, but the unique importance of bicarbonate transporters in specific cell contexts is probably only to be revealed with identification of additional genetic diseases caused by defects in bicarbonate transport.
J. R. C. is a Scientist of the Alberta Heritage Foundation for Medical Research. E. C. is the AMGEN Western Canadian Kidney Senior Research Fellow.
Members of the J. R. C. laboratory provided helpful comments on the manuscript. We thank bicarbonate transport researchers for making this review possible and apologize for any citations we have omitted.
Abbreviations: AE, Cl−/HCO3− anion exchanger; AE3c, AE3 cardiac; AE3fl, AE3 full length; AQP, aquaporin; CA, carbonic anhydrase; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CHED2, congenital hereditary endothelial dystrophy type 2; CSF, cerebrospinal fluid; eAE1, erythrocyte AE1; ER, endoplasmic reticulum; FECD, Fuch's endothelial corneal dystrophy; GABA, γ-aminobutyric acid; GPA, glycophorin A; HEK, human embryonic kidney; HS, hereditary spherocytosis; kAE1, kidney AE1; MAPK, mitogen-activated protein kinase; MDCK, Madin–Darby canine kidney; NBC, Na+/HCO3− co-transporter; NHE, Na+/H+ exchanger; pHi, intracellular pH; PKC, protein kinase C; RBC, red blood cell; RT, reverse transcription; RTA, renal tubular acidosis; dRTA, distal RTA; pRTA, proximal RTA; SAO, Southeast Asian ovalocytosis; SCFA, short-chain fatty acid; SLC, solute carrier; STAS, sulfate transporter and anti-σ factor antagonist; WT, wild-type
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