Introduction

The SLC26 anion exchangers are a relatively young gene family of highly versatile anion exchangers, with intriguing roles in normal physiology and human pathophysiology. A partial list of physiological processes in which these exchangers play critical roles includes skeletal development [27], synthesis of thyroid hormone [21], transepithelial Na+-Cl− transport [31, 42, 102], bicarbonate excretion by the distal nephron [75], and bicarbonate secretion by the exocrine pancreas [44].

SLC26 exchangers transport an expanding number of monovalent and divalent anions, including sulfate (SO4 2−), chloride (Cl−), iodide (I−), formate, oxalate, hydroxyl ion (OH−), and bicarbonate (HCO3 −) [6, 35, 38, 61, 78, 81, 87, 102]. Individual paralogs differ significantly in anion specificity, such that SLC26A6 is capable of transporting all of the substrates above [35, 102]. In contrast, SLC26A4 transports monovalent anions such as Cl−, I−, and formate, but not divalent anions such as SO4 2− and oxalate [81, 82]. Several paralogs function in Cl−-OH− and Cl−-HCO3 − exchange, with increasingly important roles in transepithelial Na+-Cl− and HCO3 − cotransport. Where direct comparisons have been made, the SLC26 proteins appear to be more potent exchangers of Cl− with HCO3 − than with OH− [44, 100, 102]. How the transport characteristics and anion specificities of the individual paralogs might contribute to their respective physiology is reviewed in the appropriate sections below.

Although this review is restricted to a discussion of the mammalian SLC26 exchangers, it should be noted that they are a part of the large "sulfate permease" or SulP family, with identifiable homologs in bacteria, plants [29, 90], fungi [11, 93], and other animals [76]. For example, the genome of the plant Arabidopsis contains 14 SLC26 homologs [29], some of which play a crucial role in sulfur assimilation from the environment [90]. Sulfate uptake via SLC26 homologs in the mold Penicillium chrysogenum in turn plays an important role in the biosynthesis of penicillin by this species [93].

Structural features

One feature of the SLC26 proteins is the relatively low conservation between orthologs in mouse and man; the percent amino acid identity ranges from a low of 76% (SLC26A8) to a high of 95% (SLC26A5), versus the reported median of 86% for mouse and human orthologous genes [55]. The HUGO nomenclature is used here; "SLC26A-" denotes a human gene/protein, "Slc26a-" denotes a rodent ortholog, and where appropriate the genes per se are written in italics. Although clearly homologous, the SLC26 proteins in Drosophila and Caenorhabditis elegans are only 25–40% identical to the mammalian proteins, such that one cannot in most instances discern orthologs for the individual mammalian exchangers. All ten of the mammalian SLC26 proteins (see Fig. 1) predict a hydrophobic core, for which the details of membrane topology are difficult to predict. However, given that the amino- and carboxy-terminal domains of SLC26A5 and SLC26A6 are intracellular [54, 104], it is expected that these proteins span the plasma membrane from 10 to 14 times [61, 76] (see Fig. 2).

Fig. 1.
figure 1

Phlylogenetic tree of the SLC26 gene family. With the exception of murine Slc26a5 (Prestin), the human orthologs were used to generate a phylogenetic tree with the Clustal W program. The percentage identity varies from 21% to 43%

Full size image
Fig. 2.
figure 2

Predicted topology of the Slc26a6 anion exchanger. The detailed membrane topology of the SLC26 exchangers has not been determined experimentally, and prediction programs yield highly divergent models. However, since the amino- and carboxy-terminal domains of SLC26A5 and SLC26A6 are intracellular [54, 104], the SLC26 proteins likely span the plasma membrane from 10 to 14 times [61, 76]. The topology shown here for murine Slc26a6 was derived using HMMTop (www.enzim.hu/hmmtop/). The position of various conserved motifs and domains is depicted, in addition to the amino-terminal extension found in the Slc26a6a protein [102] (see text for details)

Full size image

Much of the homology between SLC26 exchangers is found within the hydrophobic core of transmembrane (TM) domains. One region of homology encompasses the 22 amino-acid "sulfate transport" consensus signature (Prosite, PS01130), which was initially defined by the comparison of the first mammalian family members with homologs in lower organisms (Fig. 2). Although not all members of the mammalian family conform to the exact consensus sequence, this region contains several invariant residues which are presumably critical for anion transport. There is a second cluster of invariant residues at the C-terminal end of the hydrophobic core of the proteins, in a conserved segment defined by Saier et al. [76] (see Fig. 2). This region includes the triplet -NQE-, residues 417–419 of Slc26a2, which is conservatively variable only in Slc26a8 (-NQD-). Three highly conserved or invariant residues in this section, E419, N425, and L483 in Slc26a2, have been shown to have functional significance in SHST1, an SLC26 homologue from the plant Sporobolus hamata [40]. Moreover, two of these invariant residues are mutated (N425D and L483P) in patients with a severe defect in human SLC26A2, causing achondrogenesis type 1B and/or atelosteogenesis type 2; the SLC26A2 N425D mutant has been shown to be non-functional in Xenopus oocytes [36].

Many of the SLC26 proteins end with a class I PDZ interaction motif [88]; the exceptions include human SLC26A1 (mouse and rat Slc26a1 end with S-A-L, a typical class I motif), SLC26A2, SLC26A4, SLC26A5, and SLC26A11. These C-terminal motifs are dispensable for transport function in Xenopus oocytes [2, 54], but presumably tether SLC26 exchangers to regulatory proteins and other transporters, including NHE3 and CFTR [1, 44, 54]. The C-terminal cytoplasmic domain of all ten SLC26 proteins includes the sulfate transporter and anti-sigma (STAS) domain (Fig. 3), recently uncovered due to the homology between the SLC26 gene family and bacterial anti-sigma factor antagonists. The physiological and/or mechanistic roles of the STAS domain in the SLC26 exchangers are completely unknown; however the existence of disease-associated mutations in this domain underscores its potential importance [3]. Structural features have been predicted from the NMR analysis of the anti-sigma factor SPOIIAA, and include a characteristic α-helical handle. There is also a highly conserved loop interspersed between a β-pleated sheet and an α-helical region, just upstream of the α-helical handle. This loop and β-pleated sheet have been proposed to play a role in nucleotide binding and hydrolysis, by extension from the known biochemistry of the anti-sigma factor antagonists [3]. The loop is highly conserved in the ten SLC26 proteins and contains two invariant residues, D660 and L667, in the Slc26a2 protein. The STAS domain also contains a highly variable loop just proximal to the β-pleated sheet and putative nucleotide binding loop. This variable loop is the site of significant insertions in the SLC26 proteins, of as much as 150 amino acids in the case of SLC26A8. No such insertion is present in bacterial SLC26 homologues, and this loop is also much shorter in SLC26A11 and in the Drosophila and C. elegans paralogs; how this variable loop contributes to paralog-specific function and/or regulation is not known.

Fig. 3.
figure 3

The sulfate transporter and anti-sigma antagonist (STAS) domain. Modified from [3], with permission. The order of sequences predicted to form β-pleated sheets and α-helices is as shown. A highly conserved loop is interspersed between a β-pleated sheet and α-helix; this loop and β-pleated sheet have been proposed to play a role in nucleotide binding and hydrolysis [3]. The sulfate transporter and anti-sigma (STAS) domain also contains a highly variable loop just proximal to the β-pleated sheet and putative nucleotide binding loop. This variable loop is the site of significant insertions in the SLC26 proteins, of as much as 150 amino acids in the case of SLC26A8

Full size image

SLC26A1 (Sat-1)

Slc26a1 (Sat-1) was identified as a Na+-independent SO4 2− transporter by expression cloning from rat liver [6]. This initial report indicated that Slc26a1 functioned as a SO4 2−-anion exchanger [6]. The authors hypothesized that the likely mode of operation was SO4 2−-HCO3 − exchange, given that the heterologous transport activity mirrored sulfate exchange activity measured in liver canalicular membrane vesicles [59]. However, there is to our knowledge no direct evidence for acid-base or HCO3 − transport mediated by recombinant SLC26A1. No Mendelian diseases are known to be associated with mutations in SLC26A1—it is perhaps because of this that the current state of knowledge lags behind other established members of the gene family.

SLC26A1 has been cloned in rat [6], human [52], and mouse [102], with the predicted protein varying in size from 701 (human) to 704 (mouse) amino acids in length. Most functional studies have utilized Xenopus oocytes for heterologous expression. A consistent feature which is unique to SLC26A1/Slc26a1, demonstrated thus far for the rat and mouse orthologs [48, 78, 102], is that SO4 2− uptake by Slc26a1-oocytes is increased significantly by the presence of extracellular Cl−. Indeed, some have reported that SO4 2− uptake is essentially absent in Slc26a1 oocytes in the absence of extracellular Cl− [48, 78], whereas we have reported modest but significant SO4 2− transport under these conditions [102]. The published studies do, however, agree in the dramatic activation of SO4 2− transport by extracellular Cl−, with a reported K m of 0.136 mM (rat) [6] and 0.31 mM (mouse) [48] in the presence of extracellular Cl−. The physiological and/or mechanistic relevance of the activation by extracellular Cl− is not known. Regardless, we have recently extended this observation, showing that SO4 2− transport is activated by extracellular halides, formate, and lactate; lesser but consistent findings were found for oxalate transport [102]. Whereas we have not found that Slc26a1 oocytes transport Cl−, even in the presence of extracellular SO4 2−, Lee et al. see a modest increase in Cl− uptake in Slc26a1-injected oocytes [48]. The available data suggest that the basolateral SO4 2− exchanger of the proximal tubule (Slc26a1, see below) also does not transport Cl− [46]. Finally, SO4 2− exchange by Slc26a1 is significantly stimulated by an acid-outside pH gradient, particularly in the presence of extracellular Cl− [102]; it is as yet unclear whether this activation occurs because co-transported H+-SO4 2− are together exchanged with intracellular anions, as reported for AE1 [12], or because SO4 2−-OH− exchange is a dominant mode for Slc26a1.

Despite the cloning of Slc26a1 as the hepatic canalicular SO4 2−-anion exchanger, its physiological role in this tissue is not to our knowledge completely clear. However, the functional characteristics match basolateral anion exchange activities reported for the renal proximal tubule of both mammals [46, 67] and teleosts [71], and expression of the Slc26a1 protein has been localized to the basolateral membrane of this nephron segment [38]. Given the ability of Slc26a1 to transport oxalate [38, 102], it likely participates with apical Slc26a6 (see Fig. 4) in secretion of this anion by the proximal tubule. In addition to liver and kidney, Slc26a1 transcript is detectable in calvaria, brain, heart, and skeletal muscle [48]; physiological roles in these tissues are as yet unknown.

Fig. 4A, B.
figure 4

Transepithelial Na+-Cl− absorption in the renal proximal tubule. SLC26A6 is the leading candidate for the apical Cl− entry site, functioning as either Cl−-base exchanger or as a Cl−-formate/oxalate exchanger (A). Simplified model showing Na+-Cl− transport via coupled Cl−-base exchange and Na+-H+ exchange. Na+ and Cl− exit the cell via K+-Cl− cotransport, Cl− channels, and the Na+-K+-ATPase. B Na+-Cl− absorption at the apical membrane via either Cl−-oxalate exchange coupled to SO4 2− transport or Cl−-formate and formate-OH− exchange coupled to Na+-H+ exchange

Full size image

SLC26A2 (DTDST)

SLC26A2 was identified via positional cloning of the gene for diastrophic dysplasia (OMIM #222600), on chromosome 5q32 [27]. The name for this syndrome is derived from the geological term "diastrophism," referring to the bending of the earth's crust to form geological features. The most prominent feature of diastrophic dysplasia is a normocephalic, short-limbed dwarfism; however, these patients can also have a significant number of skeletal defects, including cleft palate, kyphoscoliosis, and clubbed feet. Soon after the characterization of SLC26A2, recessive loss-of-function mutations were reported in other recessively-inherited chondrodysplasias including achondrogenesis 1B (ACG-1B), a severe skeletal dysplasia results in prenatal or early neonatal death [28, 89]. Mutations in SLC26A2 have more recently been reported in patients with milder defects [4] and/or features that are somewhat atypical for diastrophic dysplasia [58], emphasizing the wide spectrum of associated disease.

The SLC26A2 gene product, aka DTDST (diastrophic dysplasia sulfate transporter), is some 739 amino acids in length, with ~45% identity to the SLC26A1 protein. The SLC26A1 and SLC26A2 genes share a similar organization, with the bulk of coding sequence contained within one exon [102]. Expression of human SLC26A2 and rat Slc26a2 in Xenopus oocytes results in robust SO4 2− uptake that is cis-inhibited by extracellular Cl− or oxalate and sensitive to 1 mM DIDS [78]. Direct studies of substrates other than SO4 2− are lacking and thus the full range of anions transported by SLC26A2 is not known; however, given the ability of both monovalent and divalent anions to cis-inhibit, it is likely to transport multiple anions. The correlation of clinical phenotypes with functional characterization of disease-associated mutations in Xenopus oocytes reveals a rough phenotypic series; ACG-1B is typically associated with homozygous transport-null mutations, whereas mutations causing milder phenotypes result in significant residual SO4 2− uptake [36].

Although particularly abundant in intestine and cartilage [78], varying amounts of SLC26A2 transcript are detectable in most tissues [26, 27]. Immunohistochemistry detects SLC26A2 protein in chondrocytes of developing fetal hyaline cartilage and fetal bone [26], and murine Slc26a2 is induced by bone morphogenetic protein-2 during the in vitro differentiation of a fibroblast cell line into osteoblasts [45]. SLC26A2 protein is also expressed at the apical membrane of colonic epithelial cells, in placental trophoblasts, eccrine sweat glands, and bronchial submucosal glands [26]. The widespread expression of SLC26A2 is somewhat surprising, given the specific involvement of this exchanger in chondrodysplasias; presumably anion transport by other paralogs is able to compensate for deficits in SLC26A2 in tissues other than cartilage and bone. Homozygous L483P loss-of-function [36] mutations in SLC26A2, detected in a patient with ACG-1B, do however result in a marked decrease in SO4 2− uptake in the patient's fibroblasts and chondrocytes, accompanied by marked undersulphation of cartilage proteoglycans [73]. Inhibition of proteoglycan sulphation has also been shown to reduce the proliferative response of rat chondrocytes to basic fibroblast growth factor-2 [78], an important skeletal morphogen [15, 85]. Sulphation of proteoglycan is required for multiple developmental cues, such that defects in the various transport and enzymatic pathways involved can have profound developmental consequences [64]. It is thus not surprising that defective SO4 2− transport by SLC26A2 causes such a spectrum of abnormalities in skeletal development; whether SO4 2− transport by other SLC26 exchangers plays a similar developmental role is an intriguing issue.

SLC26A3 (DRA, CLD)

SLC26A3 was initially identified as a potential tumor suppressor that was down-regulated in adenoma (hence the alias DRA) and abundant in normal colonic mucosa [80]. Downregulation of SLC26A3 in neoplastic cells is attributed more to its status as a marker of differentiated colonic epithelium than to a role in cellular proliferation [7, 86]. Although the risk of colonic malignancy in kindreds with mutations in SLC26A3 is not dramatically increased [30], a recent report revealed that inducible over-expression of the SLC26A3 protein leads to growth suppression of cultured cell lines [10], hence a role in the regulation of cell growth has not been completely ruled out.

The SLC26A3 protein is most similar to SLC26A4, with 44% identity and 60% similarity. Reports that SLC26A3 transports both SO4 2− and oxalate when expressed in Xenopus oocytes [86] are thus somewhat surprising, given that SLC26A4 transports only monovalent anions [81]. Despite evidence that SO4 2− and oxalate transport by SLC26A3 is DIDS-sensitive and mutually cis-inhibitory, the absolute values (in pmol/oocyte/h) in these two studies [86] are as much as several hundred-fold lower than those reported for other members of the family [36, 81, 82, 102]. A quantitative re-evaluation of this issue would be of considerable interest, comparing SO4 2− and oxalate transport in SLC26A3-injected oocytes to the appropriate controls (e.g., Slc26a6 oocytes [35, 102]) for these anions. That being said, moderate SO4 2− transport by Sf9 armyworm, Spodoptera frugiperda cells expressing SLC26A3 has also been reported [8].

The evidence that SLC26A3 functions as a Cl−-base exchanger is a good deal more convincing, coming as it does from several transport physiology laboratories [2, 44, 60]. Indirect evidence that Cl−-HCO3 − mediated by murine Slc26a3 is electroneutral [60] appears to have been misleading, since direct measurement unveils significant changes in membrane voltage in response to HCO3 − and Cl− addition or removal [44]. Changes in membrane voltage under equivalent conditions are opposite to those seen in SLC26A6 oocytes, indicating that the stoichiometries of these two exchangers are opposite. The relevance of this physiology to HCO3 − transport is discussed in the SLC26A6 section below.

SLC26A3 transcript is predominantly expressed in the digestive system, where it is particularly abundant in duodenum and colon [34]. SLC26A3 is expressed at the apical membrane of both surface and crypt cells in the colon [26, 61, 68]. Whereas SLC4A1 (AE1) transcript in the colon varies with salt intake and aldosterone, SLC26A3 expression is not affected [68]. Cl−-base exchange via SLC26A3 is thought to function in transepithelial salt transport by the colon, in collaboration with Na+-H+ exchange mediated by NHE3 [79]. Recessive loss-of-function mutations in the SLC26A3 gene result in severe congenital chloride diarrhea, CLD (OMIM: 21470 and 126650) [31]. The majority of cases come from three geographic areas, Finland, Poland, and Arab countries; distinct founder effect mutations have been characterized from all three regions [56]. Loss of transport function has been reported for several mutations by Moseley et al. [61], with the caveats discussed above. More recently, Ko et al. demonstrated that the mutant V310del murine Slc26a3 protein, equivalent to the human Finnish founder effect mutation, is trapped within the endoplasmic reticulum, leading to a non-functional Cl−-base exchanger [44]. Regardless, the involvement of SLC26A3 in CLD indicates that this exchanger is crucial for the absorption of Na+-Cl− from the colon. Unlike SLC26A2 [36] and SLC26A4 [83], a detailed comparison of the multiple SLC26A3 mutations reported in CLD [56] has not been published. However, it seems unlikely that an equivalent phenotypic series will be uncovered, since even affected siblings with identical mutations can have divergent clinical phenotypes [32].

SLC26A4 (Pendrin)

SLC26A4 (Pendrin) was cloned by positional characterization of the gene for Pendred syndrome (OMIM #274600) [17, 84], the most common hereditary cause of syndromic deafness. Pendred syndrome is an autosomal recessive syndrome comprising sensorineural deafness and an enlarged thyroid (goiter), sometimes accompanied by hypothyroidism [69]. Biochemical studies of an involved thyroid suggested a defect in organification of iodide in the thyroid, without impairment in iodide uptake or TSH signaling [84]; comparable insights into the associated deafness were not available prior to the cloning of SLC26A4, although patients with Pendred syndrome were known to exhibit a dilated vestibular aqueduct and/or Mondini dysplasia, a complex abnormality in cochlear structure. The SLC26A4 gene first emerged in chromosome 7-specific sequence data from the Human Genome Project, and was found to reside only 49 kb from the previously identified SLC26A3 gene [21]. In a subsequent paper, four recurrent mutations in European families with Pendred syndrome accounted for 74% of the cases, suggesting multiple founder effects [18]. The chromosomal localization of Pendred syndrome was known to be very close to that of DFNB4, an autosomal recessive form of non-syndromic deafness [17]; involvement of SLC26A4 in DFNB4 was confirmed soon after the initial cloning [50]. Many if not all patients with DFNB4 due to mutations in SLC26A4 have dilated vestibular aqueducts, as seen in Pendred syndrome [70]. Mutations in the SLC26A4 protein that are clinically associated with Pendred syndrome cause complete loss of transport function when studied in Xenopus oocytes, whereas those exclusively associated with DFNB4 have residual transport activity [83]. Although this suggests that the phenotype is largely determined by the severity of the defect in SLC26A4, other genetic and/or environmental (e.g., iodide intake) factors may also play a role [57, 91]. There is also considerable variation in vestibular phenotype in both humans and mice, suggesting other modifying influences on the inner ear physiology [23].

Human SLC26A4 expressed in both Xenopus oocytes and Sf9 cells mediates Cl− and iodide (I−) transport, exhibiting mutual cis-inhibition consistent with Cl−-I− exchange [82]. SLC26A4 appears to be selective for monovalent anions, also transporting formate but neither SO4 2− [82] nor oxalate [81]. Intracellular pH measurements in response to manipulation of bath HCO3 − and/or Cl− have demonstrated that SLC26A4 also functions as a Cl−-base exchanger [44, 87]. These functional characteristics can now be correlated with the physiological roles for SLC26A4 in thyroid and kidney, two tissues in which this gene is expressed at significant levels. SLC26A4 protein is detected at the apical membrane of thyroid follicles, facing the colloid lumen [74]. Iodide is thus taken up at the basolateral membrane by the Na+-linked I− transporter (NIS, SLC5A5) and secreted through SLC26A4 into the follicular lumen, wherein organification occurs. Presumably the expression of SLC26A4 in the mammary gland [72], endometrium, and placenta [5] reflects a similar role in iodide transport by these tissues. It seems likely that SLC26A4 is not the sole efflux pathway for I− in the thyroid, given that targeted deletion of the murine Slc26a4 gene does not result in thyroid abnormalities [23]. Moreover, the modest I− transport exhibited by the mutant SLC26A4 proteins associated with DFNB4 is evidently sufficient to maintain normal thyroid histology [83].

Considerable excitement was generated by the observation that SLC26A4 mediates Cl−-formate exchange, given possible involvement in transepithelial Na+-Cl− transport by the proximal tubule (see SLC26A6 section). However, expression of SLC26A4 in the proximal tubule is minimal or absent in several species, except perhaps for rat [87], and murine Slc26a4 is quite clearly not involved in formate-stimulated Na+-Cl− transport in this nephron segment [39]. There is however robust expression of SLC26A4 in distal type B intercalated cells [75], which are known to excrete HCO3 − via a Cl−-HCO3 − exchanger with functional similarities [19]. Targeted deletion of murine Slc26a4 reduces distal HCO3 − secretion in alkali-loaded knockout mice [75]. Along the distal nephron, SLC26A4 protein is detectable in both sub-apical vesicles and at the apical membrane, within both type B and nonA-nonB intercalated cells [41, 99]; systemic acidosis induces an intracellular shift in type B cells, whereas alkalosis increases the proportion of apical staining for SLC26A4 [97].

Finally, targeted deletion of murine Slc26a4 results in profound deafness and variable deficits in vestibular function [23]. Slc26a4 transcript is expressed throughout the endolymphatic duct and sac, suggesting a role in endolymphatic fluid reabsorption [22]. Structural abnormalities in the inner ear in Pendred syndrome/DFNB4 had been attributed in part to arrested development, and the availability of a murine model affords an important opportunity to follow changes in inner ear structure during pre- and post-natal development. The inner ear in Slc26a4-null mice is normal until embryonic day 15, at which point they begin to develop a dilated endolymphatic duct and sac. There is almost a complete absence of normal otoconia with frequent "giant" otoconia; the latter are considered a marker for biochemically abnormal endolymph in which the kinetics of otoconia dissolution and aggregation are altered [23]. In this regard, ultrastructural characterization of mitochondria-rich endolymphatic cells indicates a subtype analogous to type B intercalated cells of the distal nephron [65], suggesting that defects in SLC26A4 might be associated with marked changes in the pH of endolymphatic fluid.

SLC26A5 (Prestin)

Using a subtractive cDNA cloning strategy for messages enriched in the outer hair cell of the gerbil ear, a "molecular motor" responsible for cochlear amplification in the inner ear was identified and named Prestin (PRES/SLC26A5) [103]. Slc26a5 transcripts represented ~10% of clones differentially expressed in inner vs. outer hair cells. Although Slc26a5 is highly expressed in the ear, transcripts are detected in brain, heart, spleen, and testis [105]. The outer hair cells (OHCs) in the cochlea (inner ear) change length in response to changes in acoustic frequencies. Mammals typically respond to sound from 20 to 50,000 Hz, though the range in humans is limited to ~20 kHz [16]. This ability gives the mammalian inner ear exquisite sensitivity and frequency-resolving capacity. The transduction mechanics of this acoustic sensing is that stretch is transformed to voltage changes in the outer hair cells. Targeted disruption of mouse Slc26a5 causes "deafness," i.e., a 40- to 60-dB loss of cochlear sensitivity in the inner ear but without apparent changes in mechanotransduction [51]. Thus, the simple hypothesis is that SLC26A5 is the OHC motor molecule necessary for electromotility. Furthermore, a simple, direct coupling between cochlear amplification and electromotility exists as evidenced by Slc26a5-null mice. Human Prestin (SLC26A5) is on a human BAC clone sequence (AC005064) which maps to chromosome 7q31 near the locus for DFNB17 [103], hence evidence of involvement of the human gene in deafness will likely be forthcoming.

Hydropathy analysis showed that Slc26a5 is similar to other SLC26 proteins. However, differences within the conserved hydrophobic regions discussed above indicated that Slc26a5 might have altered transport properties, i.e., not merely mediating sulfate and/or anion exchange [103]. In fact, these investigators found a "gating current" and nonlinear capacitance change in cells transfected with Slc26a5 but not SLC26A4. Oliver et al. next demonstrated that this nonlinear capacitance change is mediated by intracellular halides, especially Cl− and HCO3 − [63]. Without Cl− and HCO3 −, these charge movements were not present, nor were they seen when using human SLC26A6. These capacitance changes were frequency dependent and were also found in inner hair cells, with both systems blocked by 10 mM Na+-salicylate. These investigators hypothesized that Prestin is the motor protein of inner hair cells and that intracellular Cl− and HCO3 − acts as the voltage "sensor" for the protein [63]; how these biophysical phenomena function in the sensory mechanism of hearing is not yet clear.

Examination of the rat Slc26a5 gene reveals a potential thyroid hormone response element (TRE) in the first intron upstream of the start codon [101]. The Slc26a5 TRE binds thyroid hormone receptors to mediate a triiodothyronine (T3)-dependent transactivation of the heterologous promoter. In situ hybridization and promoter studies revealed T3 responsiveness of the Slc26a5 mRNA and protein during development. This is of considerable clinical interest, given that congenital hypothyroidism is associated with deafness.

SLC26A6 (PAT-1, CFEX)

SLC26A6 was the first member of this family to be identified exclusively through database mining. Everett and Green had initially noted the existence of multiple SLC26 ESTs that were evidently derived from novel members of the family [20]; partial characterization of many of these genes was subsequently reported by Lohi et al., in addition to the cloning of a full-length human SLC26A6 cDNA [52]. Murine Slc26a6 was then identified as a primary candidate for the apical Cl−-formate exchanger of the renal proximal tubule [42]. SLC26A6 is one of the more widely expressed members of the family, with particularly abundant transcript in kidney, pancreas, intestine, heart, muscle, and placenta [42, 44, 52, 98, 102]. Significant heterogeneity appears to be generated by alternative splicing of the SLC26A6 gene, with two major alternatively spliced isoforms differing in the presence or absence of an N-terminal extension of 23 amino acids in both mouse and human [52, 98, 102]. Three other functional splice variants were recently described in human SLC26A6 [53]; it will be of considerable interest to determine whether these splicing events are conserved in other species. Immunolocalization has mostly been reported for epithelial tissues, where SLC26A6 protein is apically expressed [42, 52, 66, 100].

SLC26A6 is the most convincingly versatile anion exchanger of the family, functioning in Cl−-oxalate, SO4 2−-oxalate, SO4 2−-Cl−, Cl−-formate, Cl−-HCO3 −, and Cl−-OH− exchange when expressed in Xenopus oocytes [35, 44, 100, 102]. SLC26A6 might encode the long-elusive apical Cl− entry site involved in proximal tubular Na+-Cl− absorption (Fig. 4), since it is expressed at the apical membrane of proximal tubule cells [42] and appears to mediate the multiple modes of anion exchange that have been implicated in this process [35, 102]; the relevant background is particularly well reviewed in reference [47]. In addition to the exchange of Cl− with various anions, SLC26A6 may also function in formate-OH− exchange [77], which is required for apical formate recycling (see Fig. 4). Whether SLC26A6 is the dominant apical SLC26 exchanger in the proximal tubule awaits the characterization of Slc26a6-null mice, particularly given the evidence for multiple apical Na+-H+/OH− transporters in this cell type [13, 24]. There is considerable functional and pharmacological heterogeneity of anion exchange pathways in proximal brush border vesicles [37], consistent perhaps with underlying molecular heterogeneity.

A major recent surprise was that Cl−-base exchange mediated by SLC26 family members is electrogenic, with opposite apparent stoichiometries for SLC26A6 and SLC26A3/A4 [44, 102]. Thus, whereas SLC26A6-expressing cells hyperpolarize upon removing extracellular Cl− in the presence of HCO3 − [102], SLC26A3-expressing cells exhibit a pronounced depolarization [44]; a modest hyperpolarization is also seen during Cl−-OH− exchange mediated by Slc26a6 [102]. It appears that functional CFTR is required for the activation of SLC26 Cl−-base exchange by cyclic-AMP [44]; this observation may be of profound significance for cystic fibrosis, given the role for abnormal bicarbonate transport in this disease [14]. Epithelial HCO3 − transport has both an electrogenic component, generally ascribed to the HCO3 − permeability of CFTR itself, and an electroneutral component attributed to Cl−-HCO3 − exchange (see Fig. 5). CFTR-regulated Cl−-HCO3 − exchange appears to be electroneutral [49]; however, cyclic-AMP has no effect on Cl−-HCO3 − exchange mediated by the electroneutral SLC4 anion exchangers AE1–3 [44]. CFTR does, however, markedly activate Cl−-OH− exchange mediated by SLC26A3, SLC26A4, and SLC26A6 [44]. Like SLC26A6 [52], SLC26A3 protein is detected at the apical membrane of pancreatic ductal cells [25]. In cell membranes expressing an equal proportion of SLC26A6 and SLC26A3, one would expect net Cl−-HCO3 − exchange to be electroneutral; variation in the activity and/or ratio of these and other SLC26 exchangers would lead to net electrogenic HCO3 − transport [44]. The apparent stoichiometry suggests that SLC26A6 is best suited to HCO3 − excretion in the early portion of the pancreatic duct, with SLC26A3 (HCO3 −:Cl−≥2) better suited for generation of the low Cl− and HCO3 − concentration of the final secreted fluid [44] (Fig. 5).

Fig. 5.
figure 5

NaHCO3 secretion model of pancreatic duct epithelia. The pancreatic duct epithelium is the champion of HCO3 − secretion. All mammals are capable of secreting solutions of ~70 mM NaHCO3, but humans, cats, and guinea pigs are know to secrete up to isotonic NaHCO3 (150 mM NaHCO3). The traditional cellular model by which this secretion was performed contained a CFTR Cl− channel working in tandem with an apical Cl−-HCO3 − exchanger (anion exchanger) to move NaHCO3 into the duct. However, the SLC4 anion exchangers (AE1, AE2, AE3) do not fulfill this function. Moreover, decreasing apical [Cl−] and increased extracellular pH reduce the activity of these exchangers. Recently, it was appreciated that NaHCO3 can enter the blood side of the duct by NBCe1 (SLC4A4b, pNBC1) in addition to transmembrane CO2 movement. For the HCO3 − exit, two SLC26 transporters (A3 and A6) appear to use altered stoichiometry to insure apical HCO3 − exit; Cl−-base exchange by these transporters is regulated by CFTR [44]

Full size image

The apical expression of SLC26A3 and SLC26A6 within the gastrointestinal tract overlaps significantly, with robust expression of both in the duodenum [34, 100] and variable co-expression elsewhere. Jacob et al. have suggested that the relative surfeit of SLC26A3 transcript in duodenum compared to NHE2 and NHE3 reflects the role of this segment in secretion of HCO3 − [34]. SLC26A6 colocalizes with H+-K+-ATPase in gastric parietal cells, with dramatic attenuation of expression in gastric H + -K + -ATPase-null mice [100].

Finally, Slc26a6 has been shown to transport oxalate [35, 102], and the overall transport characteristics are very similar to those reported for the oxalate exchanger of ileal brush border [43]. This suggests a dominant role for SLC26A6 and other co-expressed SLC26 exchangers in the absorption of dietary oxalate by the small intestine, a major determinant of urinary excretion and thus the risk for renal stones [33].

SLC26A7-SLC26A11

Several other novel SLC26 paralogs have recently been described (see also Table 1). Although Lohi et al. included a gene now denoted SLC26A10 on chromosome 12 in their initial report [52], our own sequencing of EST cDNAs in both mouse and human suggests that SLC26A10 is an expressed pseudogene (D.B.Mount, unpublished data). There are now several Genbank cDNA accessions for this gene, none of which contain an uninterrupted open reading frame. Many of the novel SLC26 paralogs have tissue-specific expression patterns on Northern blot analysis; SLC26A7 in kidney [53, 95], SLC26A8 in testis [53, 92], and SLC26A9 in lung [53]. SLC26A11 is more widely expressed [94]. Expression of SLC26A8 in spermatocytes [53, 92] suggests a potential role in capacitation of sperm by extracellular HCO3 −, assuming that this exchanger functions in DIDS-sensitive Cl−-HCO3 − exchange [96].

Table 1. The SLC26 gene family
Full size table

Clearly the transport physiology of these newer paralogs has had less time to mature than that of the rest of the family, and will not be reviewed in detail here. However, modest anion transport has been reported for all four of the novel paralogs. SLC26A7, SLC26A8, and SLC26A9 reportedly transport Cl−, SO4 2−, and oxalate [53]. Of some concern, SO4 2− transport mediated by SLC26A9 is not cis-inhibited by oxalate [53], which is at least superficially inconsistent with the ability of this exchanger to mediate oxalate transport. No doubt the availability of four new members will greatly aid in the structure-function analysis of anion transport by these exchangers, once the complete spectrum of their transport characteristics are reported.

Outlook and pharmaceutical considerations

Tremendous progress has been made over the decade or so since the cloning of rat Slc26a1 [6], with the identification of a further nine family members and the demonstrated involvement of at least three in Mendelian genetic disorders. It is already apparent that many SLC26 paralogs have overlapping expression patterns and superficially similar transport function. For example, as many as three family members, SLC26A2, -A3, and -A6, that are capable (or potentially capable, in the case of SLC26A2) of Cl−-base exchange are expressed at the apical membrane of pancreatic ductal cells [25, 26, 44, 52, 102]. Base exchange by both SLC26A3 and SLC26A6 is regulated by CFTR [44], and thus both are implicated in CFTR-dependent HCO3 − secretion and pancreatitis [14]; it will clearly be a considerable challenge to unravel the roles and regulation of SLC26 paralogs in specific cell types such as this.

Despite the increasing interest in the transport properties of the SLC26 exchangers, there remain some significant gaps, discrepancies, and issues for future study. For example, what is the exact stoichiometry of all of the SLC26 exchangers that are capable of Cl−-HCO3 − exchange? In the case of SLC26A6, how does stoichiometry and electrophysiology differ in its multiple exchange modes, including divalent-divalent, monovalent-monovalent, and divalent-monovalent exchange? What are the regulatory and/or mechanistic implications of the cytoplasmic STAS domain? How does the primary structure of the SLC26 proteins confer anion specificity and/or versatility? Ultimately, one hopes that the study of bacterial and/or Drosophila paralogs of the SLC26 exchangers will include the determination of crystal structure, to help solve these important questions.

In addition to the obvious relevance of specific paralogs to the diagnosis and classification of genetic disease [36, 83], the SLC26 family is increasingly relevant to human disease. In particular, CFTR-independent activation of SLC26 Cl−-HCO3 − exchange is likely a worthy goal in cystic fibrosis [44]. Given the variability and progressive nature of the inner ear phenotype, further study of Slc26a4-null mice will hopefully suggest therapeutic targets for this and related disorders, including Ménière's disease [23]. Delineation of the critical SLC26 exchangers for intestinal absorption and renal excretion of oxalate will also help target these pathways for the prevention and/or treatment of kidney stones [102]. Finally, recent data implicate DIDS-sensitive intestinal anion exchangers, potentially members of the SLC26 gene family, in the intestinal transport of drugs such as salicylate [62] and ciprofloxacin [9].