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¹ Molecular and Vascular Medicine Unit; Renal Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Abstract

Plasmalemmal Cl^––HCO₃^– exchangers regulate intracellular pH and [Cl^–] and cell volume. In polarized epithelial cells, they contribute also to transepithelial secretion and reabsorption of acid–base equivalents and of Cl^–. Members of both the SLC4 and SLC26 mammalian gene families encode Na⁺-independent Cl^––HCO₃^– exchangers. Human SLC4A1/AE1 mutations cause either the erythroid disorders spherocytic haemolytic anaemia or ovalocytosis, or distal renal tubular acidosis. SLC4A2/AE2 knockout mice die at weaning. Human SLC4A3/AE3 polymorphisms have been associated with seizure disorder. Although mammalian SLC4/AE polypeptides mediate only electroneutral Cl^––anion exchange, trout erythroid AE1 also promotes osmolyte transport and increased anion conductance. Mouse AE1 is required for DIDS-sensitive erythroid Cl^– conductance, but definitive evidence for mediation of Cl^– conductance is lacking. However, a single missense mutation allows AE1 to mediate both electrogenic SO₄^2––Cl^– exchange or electroneutral, H⁺-independent SO₄^2––SO₄^2– exchange. In the Xenopus oocyte, the AE1 C-terminal cytoplasmic tail residues reported to bind carbonic anhydrase II are dispensable for Cl^––Cl^– exchange, but required for Cl^––HCO₃^– exchange. AE2 is acutely and independently inhibited by intracellular and extracellular H⁺, and this regulation requires integrity of the most highly conserved sequence of the AE2 N-terminal cytoplasmic domain. Individual missense mutations within this and adjacent regions identify additional residues which acid-shift pH_o sensitivity. These regions together are modelled to form contiguous surface patches on the AE2 cytoplasmic domain. In contrast, the N-terminal variant AE2c polypeptide exhibits an alkaline-shifted pH_o sensitivity, as do certain transmembrane domain His mutants. AE2-mediated anion exchange is also stimulated by ammonium and by hypertonicity by a mechanism sensitive to inhibition by chelation of intracellular Ca²⁺ and by calmidazolium. This growing body of structure–function data, together with increased structural information, will advance mechanistic understanding of SLC4 anion exchangers.

(Received 5 October 2005; accepted after revision 13 October 2005; first published online 20 October 2005)
Corresponding author S. L. Alper: Molecular and Vascular Medicine Unit, E/RW-763 Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA. Email: salper{at}bidmc.harvard.edu

Electroneutral Cl^––HCO₃^– exchange contributes to cellular regulation of intracellular pH (pH_i) and cell volume, and secondarily to membrane potential through its contribution to control of the transmembrane Cl^– gradient. Cl^––HCO₃^– exchangers are found among polypeptide products of two evolutionarily unrelated gene superfamilies, SLC4 and SLC26. The anion exchanger polypeptide products of these evolutionarily unrelated gene superfamilies exhibit similarities and differences in phylogeny, tissue distribution, anion selectivity, regulatory properties and mechanism. Deficiencies in expression of these polypeptides lead to distinctive phenotypes. This brief review will focus on electroneutral anion exchangers of the SLC4 gene family, highlighting genetics, aspects of molecular mechanism and regulation.

The AE anion exchangers among the bicarbonate–anion transporters of the SLC4 gene family

The SLC4 gene family (Alper, 2002; Romero et al. 2004) includes the Na⁺-independent, electroneutral Cl^––HCO₃^– exchangers SLC4A1/AE1,SLC4A2/AE2, SLC4A3/AE3 and (more controversially) SLC4A9/AE4. Other SLC4 gene products include electrogenic (SLC4A4/NBCe1 and SLC4A5/NBCe2) and electroneutral Na⁺–HCO₃^– cotransporters (SLC4A7/NBCn1), and Na⁺-dependent Cl^––HCO₃^– (or NaHCO₃–HCl) exchangers (SLC4A8 and, more controversially, SLC4A10). The SLC4A11/BTR1 electrogenic Na⁺–borate cotransporter (SLC4A11/BTR1; Park et al. 2004) is closest in sequence among mammalian SLC4 polypeptides to the phylogenetically oldest SLC4 transporter, the borate transporter of S. cerevisiae. Molecular cloning and functional expression of SLC4 gene products from multiple mammalian species, teleost fish (Paw et al. 2003; Shmukler et al. 2005; Guizouarn et al. 2005), marine invertebrates, insects, ascidians and the roundworm C. elegans (Sherman et al. 2005) have also been reported (Romero et al. 2004). Unlike SLC26 anion transporters, SLC4 homologues have not been detected in prokaryotic genomes.

Structure of SLC4 AE polypeptides

All SLC4 polypeptides share a common structural pattern of three domains. An N-terminal cytoplasmic domain of 400–700 amino acids is followed a C-terminal polytopic transmembrane domain of 500 amino acids and a C-terminal cytoplasmic domain of 30–100 amino acids. Most SLC4 genes use multiple promoters to generate variant 5'-transcripts encoding polypeptides with distinct N-terminal amino acid sequences. The AE1 gene encodes the longer erythroid AE1 (eAE1, historically known as ‘red cell band 3’) and the shorter kidney AE1 (kAE1), which in humans initiates at Met 66. The mouse AE2 gene encodes five N-terminal variant polypeptides, while the AE3 gene encodes two variant N-terminal and two variant C-terminal polypeptide sequences.

Most structural information comes from more than three decades of study of the native erythroid AE1 protein and from more recent mutagenesis studies of recombinant AE1. A current model of the AE1 monomer is shown in Fig. 1. AE1 (and AE2) are dimers in the membrane and in detergent solution. The extreme N-terminal sequence of human AE1 binds multiple glycolytic enzymes and haemoglobin. Other parts of the N-terminal cytoplasmic domain provide binding sites for the erythroid cytoskeletal proteins ankryin-1, protein 4.2, and the ERM protein 4.1R. The N-terminal cytoplasmic domain also contains targeting information for polarized cell expression in chicken AE1 (Adair-Kirk et al. 2003). Solution of a 2.6 Å X-ray structure of the dimeric human erythroid AE1 cytoplasmic domain (amino acids 1–379) required crystallization at pH 4.8 and resolved residues 55–201 and 212–356 (Zhang et al. 2000). This structure has since helped model the location and effects of engineered and naturally occurring mutations in corresponding regions of AE2 and NBCe1.

View larger version (45K): [in this window] [in a new window]   Figure 1.  Proposed topographical model for the human SLC4A1/AE1 Cl––HCO3– exchanger polypeptide, after Zhu et al. (2003) Met 66 (arrow) marks start of kidney AE1. Polymorphisms encoding blood group antigens are in blue. The mutations associated with hereditary spherocytic anaemia and ovalocytosis are in orange, and include missense, nonsense, splicing and deletion mutations. Mutations associated with dominant and recessive distal renal tubular acidosis are in green. Terminal deletions are in lighter orange and green (Modified with permission from Shayakul & Alper (2004)Clin Exp Nephrol8, 1–11.). Upper left: scanning electron micrographs of wild-type erythrocytes and AE1(–/–) bovine spherocytes (HS; modified from Inaba et al. 1996). Upper right: consecutive semithin sections from rat kidney cortex immunostained with antibody recognizing vH+-ATPase (left) and kAE1 (right). Note that only the Type A intercalated cell with apical vH+-ATPase expresses basolateral kAE1 (from Alper et al. 1989).

E681 of human AE1 (E699 in mouse) has been identified as part of the anion translocation pathway and the permeability barrier (Chernova et al. 1997; Jennings, 2005). Other residues have been proposed to contribute to the anion translocation pathway and the anion selectivity filter based on methanethiosulphonate modification of introduced Cys residues (Zhu et al. 2003; Zhu & Casey, 2004). The transmembrane domain includes interaction sites for erythroid glycophorin A, which acts like a ‘ß-subunit’ for AE1 trafficking and optimal function. Carbonic anhydrase IV also interacts with exofacial portions of SLC4 transmembrane domains, as may the transmembrane exo-carbonic anhydrases. The exofacial loops of AE1 also present blood group antigens to the immune system and serve as part of the Plasmodial merozoite invasion receptor (Goel et al. 2003).

The C-terminal tail of many SLC4 polypeptides contains one or more acidic motifs that constitute a core binding site for cytoplasmic carbonic anhydrase II. AE1 targeting information for expression in the Type A intercalated cell is also present in the C-terminal tail. AE1 undergoes extensive covalent modification in the cell, including N-glycosylation, palmitoylation and phosphorylation, but the functions of these modifications are unknown in mammalian AE1. Tyrosine phosphorylation of skate AE1 has been proposed to play a role in regulatory volume decrease mediated by AE1-associated osmolyte transport in skate erythrocytes (Musch & Goldstein, 2005).

Disease phenotypes of AE1 mutations and knockouts

The AE1 polypeptide is expressed in greatest abundance in erythrocytes and in the Type A acid-secreting intercalated cells of the renal collecting duct. Mutations of the human AE1 gene (Fig. 1) are characterized by erythroid and renal phenotypes (see Alper, 2003 for a list). One group of mutations (blue in Fig. 1) encodes almost entirely asymptomatic polymorphisms encoding blood group antigens recognized by patient antisera in intact erythrocytes. These have served to establish topography of the membrane domain of the AE1 polypeptide. The largest group of mutations (orange in Fig. 1) is associated with autosomal dominant hereditary spherocytic anaemia (HS, a disease also caused by mutations in spectrin and ankyrin). HS red cells are characterized by reduced surface area and osmotic fragility. Many of these mutant alleles generate unstable mRNA, and the resultant erythrocytes express reduced levels of wild-type eAE1 polypeptide. These patients have an apparently normal renal acidification phenotype.

Occasional patients with severe HS have been found to be compound heterozygotes. Only two AE1 mutations have been found in severe, early-onset recessive HS, in both cases progeny of consanguineous parents with mild autosomal dominant HS. Band 3 Neapolis (Perrotta et al. 2005) is an intron 2 splice donor site mutation resulting in skipping of exon 2 and unstable mRNA encoding an AE1 polypeptide lacking the N-terminal 11 amino acids. This mutant polypeptide, present at only 12% of the normal level, lacks the binding site for glycolytic enzymes. Homozygosity for Band 3 Coimbra (AE1 V488M) is associated with complete absence of AE1 and causes severe hydrops fetalis with haemolytic spherocytic anaemia and recessive renal tubular acidosis (RTA) (Ribeiro et al. 2000).

South-east Asian Ovalocytosis (SAO) is caused by autosomal dominant in-frame deletion of hAE1 400–408 (orange in Fig. 1). Homozygotes are presumed to be embryonic lethal. Heterozygote red cell membranes exhibit increased rigidity and cold-induced cation permeability, and the allele seems to confer protection against cerebral complications of malaria. The stable mutant polypeptide is present at normal abundance in the membrane, where it heterodimerizes at apparent normal affinity with wild-type polypeptide (Jennings & Gosselink, 1995). Although AE1 SAO is itself functionally inactive in both Cl^––Cl^– and Cl^––HCO₃^– exchange (Dahl et al. 2003), the minimal impact of its dominant effects upon the wild-type monomer within the SAO/wt heterodimer (Kuma et al. 2002; Cheung et al. 2005a) explain its lack of renal phenotype in the absence of a second mutant allele of AE1.

Distal renal tubular acidosis (dRTA) is caused by a different set of AE1 mutations (green in Fig. 1), almost entirely distinct from those causing erythroid dyscrasias and rarely accompanied by an erythroid phenotype. dRTA is characterized by impaired urinary acid excretion in the setting of metabolic acidosis or imposed acid loading, leads to growth retardation, and can be accompanied by hypercalciuria and hypokalaemia. Inadequate treatment with bicarbonate supplementation can lead to osteomalacia, nephrocalcinosis and nephrolithiasis. The lack of an intercalated cell model in tissue culture has slowed our understanding of the mechanisms of dRTA. Moreover, the overexpressed mutant polypeptides can express distinct trafficking phenotypes depending on host cell type, plating matrix, and degree of confluence and (for epithelial cells) polarization (Shayakul & Alper, 2004).

The AE1 mutants associated with dominant dRTA expressed in Xenopus oocytes usually exhibit normal or modestly reduced Cl^– and HCO₃^– transport function inadequate to explain the renal phenotype. However, these mutant polypeptides express two types of abnormalities in mammalian cells. In the case of dominant dRTA mutations, such as AE1 R589H (Jarolim et al. 1998), they are themselves retained in the endoplasmic reticulum and appear to exert a dominant negative trafficking phenotype in heterodimers with wild-type AE1. A second class of dominant dRTA mutation is exemplified by AE1901X, lacking the C-terminal 11 amino acids. This mutant accumulates in the apical as well as the basolateral membrane of polarized epithelial cells, apparently due to loss of a sorting or retrieval signal related to the residues 904–907 (Devonald et al. 2003; Toye et al. 2004). The presence of kAE1in the apical membrane of the Type A intercalated cell probably short circuits acid secretion.

AE1 mutants causing recessive dRTA are found in Thailand (Tanphaichitr et al. 1998), Malaysia and New Guinea (Bruce et al. 2000). As exemplified by recessive dRTA mutant AE1 G701D, these mutant polypeptides are retained inside the cell, but can be rescued to the cell surface by coexpression of the ‘eAE1 ß-subunit’ glycophorin A. This rescue by an erythroid protein not expressed in renal intercalated cells can explain the normal erythroid AE1 expression in these patients (Tanphaichitr et al. 1998). Recessive dRTA with ovalocytosis can also be caused by compound heterozygosity of AE1 SAO with another AE1 loss-of-function mutation (Bruce et al. 2000). Although recessive dRTA tends to be clinically more severe than the dominant form, no other genotype–phenotype correlations have yet emerged among AE1 mutations causing dRTA.

The AE1(–/–) mouse has combined severe haemolytic anaemia (Peters et al. 1996) and a hypercoagulable state (Hassoun et al. 1998), accompanied by dRTA caused by reduced Type A intercalated cell Cl^––HCO₃^– exchange activity (Stehberger et al. 2004). A bovine cohort presenting with hydrops fetalis (Inaba et al. 1996) and the anaemic zebrafish retsina mutants are also AE1 loss-of-function mutations. Erythroid precursors in both zebrafish and mouse reveal a cytokinesis defect morphologically resembling that in human congenital dyserythroipoietic anaemia Type II (Paw et al. 2003), although band 3 mutations have yet to be found in these latter patients.

Disease phenotypes associated with SLC4A2/AE2 and SLC4A3/AE3 gene products

AE2 is the most widely expressed of the electroneutral SLC4/AE anion exchangers, present at highest levels in the epithelial cell basolateral membrane of choroid plexus, gastric parietal cells, throughout the GI tract, and in some cell types of the respiratory and genital tracts. AE2 in exocrine glands is expressed in acinar cell basolateral membranes, but minimally in duct epithelial cells. It is also expressed throughout the nephron, but most abundantly in the medullary thick ascending limb and the inner medullary collecting duct. AE2 mRNA has been found in osteoclasts, where it may function as a basolateral Cl^––HCO₃^– exchanger. Subapical (Tietz et al. 2003) or apical immunolocalization of AE2 (Aranda et al. 2004) has been observed in hepatobiliary epithelial cells. However, a mechanistic basis for the targeting differences between biliary and other epithelial cell types remains unclear.

No hereditary human diseases have been mapped to the AE2 gene. The AE2(–/–) mouse suffers severe growth retardation and dies at or before weaning. It displays failure of tooth eruption and gastric achlorhydria, along with a mild gastric mucosal dysplasia (Gawenis et al. 2004). This severe phenotype contrasts interestingly with the much milder phenotype of a mouse engineered to lack AE2a, AE2b1 and AE2b2. This grossly normal mouse shows only male infertility associated with testicular dysplasia (Medina et al. 2003). Neither expression status of AE2c1, normally limited to gastric mucosa, nor possible compensatory upregulation of other SLC4 or SLC26 anion exchangers was reported.

SLC4A3/AE3 is most abundantly expressed in brain and heart, but is also present in gut epithelial cells. Although blot-competent antibodies are available, no published antibodies have yet demonstrated histological immunospecificity tested against AE3(–/–) tissue (Alper SL & Shull GE, unpublished observations). Human AE3 mutations have not been linked to disease, but the AE3 A867D polymorphic variant has been found with elevated frequency among patients with idiopathic generalized epilepsy (Sander et al. 2002).

Mechanism of electroneutral anion exchange by SLC4/AE polypeptides

SLC4A1/AE1 mediates 1 : 1 electroneutral exchange of a wide range of monovalent anions, but Cl^– and HCO₃^– are the physiological substrates. Red cell AE1 has also been shown to mediate H⁺–sulphate and H⁺–oxalate cotransport in electroneutral exchange for Cl^–. Exchange can be modelled by ping-pong kinetics, with alternating access of substrate binding sites and with modifications for a second, possibly regulatory anion binding site. The equilibrium distribution of inward-facing and outward-facing conformations is anion substrate dependent, but this distribution is not thought to be physiologically important (Knauf & Pal, 2003; Jennings, 2005).

Binding of carbonic anhydrase II to the C terminal cytoplasmic tail of SLC4A1/AE1 has been shown to constitute a ‘transport metabolon’ in HEK 293 cells, providing or dissipating transport substrate near the anion transport site as required. The concept was subsequently extended to exofacial interaction of AE1 and carbonic anhydrase IV (Sterling et al. 2002), and to other SLC4 bicarbonate transporters. The functional importance of this transport metabolon has not been proved in the intact red cell, but accruing evidence supports its importance in recombinant expression systems. In HEK 293 cells, dominant negative carbonic anhydrase coexpression inhibits SLC4A/AE-mediated Cl^––HCO₃^– exchange. More dramatically, in Xenopus oocytes, AE1 mutations interfering with carbonic anhydrase II binding abolish AE1-mediated Cl^––HCO₃^– exchange with no inhibition of Cl^––Cl^– exchange (Dahl et al. 2003). Thus, bound carbonic anhydrase can act as an essential subunit for SLC4-mediated Cl^––HCO₃^– exchange, but is dispensable for other modes of Cl^––anion exchange.

Expression in Xenopus oocytes of trout AE1 is associated with increased constitutively active Cl^– conductance and osmolyte permeability. The Cl^– conductance has been mapped to a combination of two discreet regions of the trout AE1 transmembrane domain, and in engineered mutants need not be tightly linked to the anion exchange mechanism. Oocyte osmolyte transport associated with skate AE1 expression requires activation by hypotonic swelling (Koomoa et al. 2005), unlike trout AE1. In contrast, expression in Xenopus oocytes of AE1 polypeptides from mouse, zebrafish or skate (Guizouarn et al. 2005), or of AE2 polypeptides from mouse or zebrafish (Shmukler et al. 2005) does not increase Cl^– conductance.

Optional electrogenic anion exchange has been generated in human and mouse AE1 by modification of a Glu residue at the inner face of putative transmembrane span 8. Chemical modification of human AE1 E681 to hydroxynorvaline or mutation of mouse E699 to glutamine creates transporters which mediate electrogenic 1 : 1 exchange of internal SO₄^2– for extracellular Cl^–. Mouse AE1 E699Q can also mediate electroneutral sulphate homoexchange, but cannot mediate detectable efflux of intracellular Cl^– in exchange for any extracellular anion. Sulphate transport by these mutant AE1 polypeptides, whether electrogenic or electroneutral, is unaccompanied by H⁺ cotransport. These properties together suggest that Glu 681/699 serves as the H⁺ binding site during H⁺–SO₄^2– cotransport and as part of the permeability barrier within the AE1 anion translocation pathway (Chernova et al. 1997; Jennings, 2005).

Chemical modification of AE1 in red cells has suggested that, in addition to glutamate residues, histidine, arginine and lysine residues each contribute to anion transport and selectivity. Systematic introduction of individual cysteine residues into the Cys-less transmembrane domain of AE1 has identified several additional regions which, when reacted with sulphydryl modifiers, can alter transport function (Zhu & Casey, 2004; Zhu et al. 2003).

Erythroid Cl^– conductance is central to control of red cell volume during passage through capillaries, during oxidative stress of haemoglobinopathies and during intraerythrocytic replication of plasmodia. The DIDS sensitivity of a portion of the basal erythroid Cl^– conductance has long suggested that AE1 might mediate this conductance, perhaps by a ‘tunnelling’ mechanism.

The DIDS-sensitive component of anion conductance is indeed lacking in murine AE1(–/–) erythrocytes, but many other membrane proteins are also reduced in abundance in these fragile spherocytic cells. Thus, although AE1 expression is required for expression of erythroid Cl^– conductance, that conductance may still be mediated by a distinct ion channel polypeptide (Alper et al. 1998).

Acute regulation of SLC4/AE-mediated anion exchange

As regulators of cellular and systemic pH, SLC4/AE anion exchangers might be expected to be sensitively regulated by pH. However, this is not true in the physiological pH range for all SLC4 anion exchangers. Erythroid AE1 needs, under all conditions, to move CO₂ from the acidic environment of respiring tissues to the lungs for exhalation. Thus, SLC4A2/AE2 expressed in Xenopus oocytes exhibits several modes of acute regulation that are absent or attenuated in SLC4A1/AE1, including inhibition by protons, activation by hypertonicity and activation by NH₄⁺.

Intracellular and extracellular protons each inhibit AE2-mediated Cl^– exchange by independent mechanisms. pH_i is changed independently by isohydric addition and removal of the permeant weak acid butyrate, itself neither a transport substrate nor competitive inhibitor of SLC4/AE polypeptides. pH_o is regulated independently by bath pH change during butyrate clamp of oocyte pH_i (Stewart et al. 2002). The independent inhibitory effects of pH_o and pH_i on AE2-mediated Cl^– exchange are evident within the physiological range, but inhibition of AE1 in oocytes requires extracellular proton concentrations nearly 100-fold higher. Whereas the pH_o(50) value for AE2 (pH at which maximal Cl^– efflux is 50% inhibited) is 6.8 and that for AE1 5.0; exposure to 40 mM butyrate, lowering pH_i from 7.3 to 6.8, inhibits AE2 by 80–90%, but AE1 remains completely uninhibited. The sigmoidal pH dependence of AE2 Cl^– transport in the physiological pH range requires the AE2 transmembrane domain. Ongoing attempts to delineate specific segments of the transmembrane domain indicate that substitution of any region with the corresponding segment of pH-insensitive AE1 attenuates pH sensitivity of AE2. Evaluation of individual transmembrane domain His residues indicates that they play a cooperative role in their contribution to pH sensitivity, but do not suffice to control the full response. Most charged residues of the transmembrane domain can also be individually neutralized without altering either basal transport activity or the independent inhibition by acidic pH_o or pH_i. However, one small region of the AE2 transmembrane domain corresponding to a proposed re-entrant loop structure in AE1 appears to play an important role in anion transport regulation by both pH_o and pH_i (Stewart AK et al. unpublished observations).

AE2 residues of the intracellular N-terminal cytoplasmic domain also contribute to setting the pH sensitivity of anion transport. Mutation of some residues alters responses to both pH_o and pH_i, whereas mutation of others alters only one or the other response. Of particular note is the physiological AE2 variant polypeptide AE2c1, which lacks the N-terminal 199 amino acids of the longest of the five murine AE2 polypeptides, AE2a. The pH_o(50) of AE2c1 is 7.7, compared to 6.8 for AE2a. The basis for this difference is found in two groups of AE2a residues within amino acids 120–150 of the region missing from AE2c1. In contrast, responses of the two AE2 isoforms to acidic pH_i are indistinguishable. The pH_o and pH_i responses of the AE2 isoforms AE2b1 and AE2b2 do not differ from that of AE2a. AE2c1 is predominantly expressed in the gastric parietal cells which alkalinize the mucosal interstitial fluid during stimulated gastric acid secretion. The expression in the parietal cell basolateral membrane of AE2 polypeptide variants with overlapping pH_o sensitivities serves to broaden the pH_o range over which parietal cell basolateral Cl^––HCO₃^– exchange can be regulated by pH_o, while allowing other mechanisms of pH_i homeostasis (Kurschat et al. In press).

Several regions of the N-terminal cytoplasmic domain shared by all AE2 polypeptide variants have been characterized as critical for pH regulation of anion exchange activity. Among these is the N-terminal cytoplasmic domain sequence most highly conserved among the entire SLC4 gene family, including among the Na⁺-dependent HCO₃^– transporters, AE2a 336–347. Within this stretch, Ala substitution of three residues selectively abolishes regulation by pH_i without altering regulation by pH_o. Additional residues in the region between AE2a amino acids 200 and 500 have been shown to contribute to the independent regulation of anion transport rate by pH_o and by pH_i. Together, these residues are modelled to form semicontiguous patches on the surface of the AE2 N-terminal cytoplasmic domain (Fig. 2; Stewart AK et al. 2004; and unpublished observations).

View larger version (46K): [in this window] [in a new window]   Figure 2.  Model of mouse AE2a NH2-terminal cytoplasmic domain amino acids 317–623, highlighting conserved residues required for normal regulation of Cl––anion exchange by pHo and pHi A, ribbon diagram structure of AE2 amino acids 317–623 based on crystal structure of the corresponding region of human AE1 (Zhang et al. 2000). The structural model (above) and the linear schematic (below) each indicate residues which, when mutated, alter AE2 regulation by pHi (yellow), by pHo (red) or by both pHi and pHo (orange). B, space-filling structure of AE2 amino acids 317–610, with surface amino acid residues marked by the same colour scheme. P610 (blue) is the most C-terminal surface residue in this view. AE2 amino acids 403–408 are at the bottom in pink; mutation en bloc altered sensitivity only to pHo, but individual mutations with the same effect have yet to be identified. AE2 amino acids 397–402 are located out of view on far side at the bottom right, adjacent to amino acids 403–408. L323 is modelled to be not at the domain surface. C, the indicated sequences of mouse AE2a are aligned with corresponding regions of other SLC4 anion transporters. Boxes mark conserved residues whose mutation alters regulation of AE2-mediated Cl– transport by pH, with colour code as in A (modified from Stewart et al. 2004).

Activation of AE2 both by hypertonicity and by ammonium (accompanied by opposing changes in pH_i) requires the conserved cytoplasmic domain residues 336–347. Both activating stimuli are inhibited by chelation of intracellular Ca²⁺ and by the anticalmodulin drug, calmidazolium. However, this effect is not mimicked by calmodulin-kinase inhibitors or by other calmodulin modifier drugs. A role for other diffusible second messengers or for protein phosphorylation in the acute regulation of AE2 remains to be defined. AE2-mediated Cl^––HCO₃^– exchange is stimulated by serum in HEK 293 cells. In Xenopus oocytes, AE2 is inhibited by protein kinase C activation with phorbol ester, but this inhibitory effect has yet to be distinguished from large-scale oocyte membrane endocytosis induced by protein kinase C. It remains possible that acute regulation of AE2 activity is achieved in part or in full by control of trafficking. Tests of this mechanism should be aided by newer epitope-tagged and GFP–AE2 fusion polypeptides.

Conclusion

Understanding of the pathophysiology and genetics of SLC4 anion exchangers is gradually yielding to study of families with the AE1 diseases of hereditary spherocytosis and distal renal tubular acidosis, and to study of a growing range of knockout models. In the absence of linked human disease, elucidation of AE2 and AE3 pathophysiology will progress through investigation of animal knockout models. Ever more extensive structure–function studies, in tandem with investigation of regulatory pathways and more detailed studies of permselectivity, will continue to build a picture of SLC4 anion exchange mechanisms. Present unavailable high resolution structural information for the SLC4 transmembrane domain, as well as for much of the cytoplasmic domains, would greatly accelerate this progress.

Bruce LJ et al. (Nat Genet 37:1258-1263) have reported five heterozygous missense mutations in exon 17 of SLC4A1/AE1 in unrelated cases of hereditary Stomatocytic anemia with cation leak. These AE1 polypeptide mutants expressed alone in Xenopus oocytes are associated with reduced room temperature C1^– uptake and enhanced (but extremely low level) cation uptake at 0°C

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Acknowledgements

Thanks to The Physiological Society for its support of this Symposium; to my colleagues Andrew Stewart, Jeff Clark, David Vandorpe, Neera Dahl, Marina Chernova and Lianwei Jiang for their contributions to this work supported by NIH grant DK43495; and to Richard Vaughan-Jones for his fruitful collaboration. Constraints on article length and referencing do not permit citation of many important primary sources by valued colleagues.

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