Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains

Hannes Lohi1, Georg Lamprecht2, Daniel Markovich3, Anders Heil2, Minna Kujala1, Ursula Seidler2, and Juha Kere1,4,5

1 Department of Medical Genetics, Biomedicum Helsinki, and 4 Finnish Genome Center, University of Helsinki, 00014 Helsinki, Finland; 2 Department I of Medicine, Eberhard-Karls University, Tübingen, Germany; 3 Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia; and 5 Department of Biosciences, Novum, Karolinska Institute, 14157 Huddinge, Sweden


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The solute carrier gene family SLC26 consists of tissue-specific anion exchanger genes, three of them associated with distinct human recessive disorders. By a genome-driven approach, several new SLC26 family members have been identified, including a kidney- and pancreas-specific gene, SLC26A6. We report the functional characterization of SLC26A6 and two new alternatively spliced variants, named SLC26A6c and SLC26A6d. Immunofluorescence studies on transiently transfected cells indicated membrane localization and indicated that both NH2- and COOH-terminal tails of the SLC26A6 variants are located intracellularly, suggesting a topology with an even number of transmembrane domains. Functional expression of the three proteins in Xenopus oocytes demonstrated Cl- and SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> transport activity. In addition, the transport of SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and Cl- was inhibited by DIDS and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. We demonstrated also that the COOH terminus of SLC26A6 binds to the first and second PDZ domains of the Na+/H+ exchanger (NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor (NHERF) proteins in vitro. Truncation of the last three amino acids (TRL) of SLC26A6 abrogated the interaction but did not affect transport function. These results demonstrate that SLC26A6 and its two splice variants can function as anion transporters linked to PDZ-interaction pathways. Our results support the general concept of microdomain organization for ion transport and suggest a mechanism for cystic fibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulation in pancreatic duct cells.

alternative splicing; PDZ domain; SLC26


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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THE SYSTEMATIC CHARACTERIZATION of gene families with genome sequences provides a rich source for expanding our physiological understanding of body functions. Recently, a novel tissue-specific anion transporter gene family, SLC26, has been delineated. The members of the SLC26 family are structurally well conserved across different species and demonstrate remarkable functional similarity with the well-characterized but structurally distinct anion exchanger (AE) gene family SLC4 (2, 12). Particular interest in the SLC26 gene family is stimulated by the fact that the SLC26A2, SLC26A3, and SLC26A4 genes have been recognized as the disease genes mutated in diastrophic dysplasia, congenital Cl- diarrhea, and Pendred syndrome, respectively (5, 8, 9). Thus the three closely related but highly tissue-specific human anion transporters play central roles in the etiology of phenotypically very different recessive diseases.

In mammals, nine tissue-specific genes have been characterized in this family, namely, SLC26A1-A9. The members of the SLC26 family transport with different specificities the Cl-, I-, SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, OH-, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and oxalate anions (3, 8, 17, 18, 22, 26-29, 31, 32). SLC26A5 (prestin) was shown to act as the motor protein of cochlear outer hair cell (41, 24). More recently, SLC26A7-A9 were shown to function as tissue-specific AEs in human kidney, male germ cells, and lung, respectively (16). SLC26A8 (testis-specific anion transporter 1, Tat1) was also linked to Rho GTPase signaling (35).

SLC26A6 is structurally highly homologous to the other family members, suggesting an anion transport function (15). SLC26A6 encodes an integral membrane protein with a predicted 10 or 12 transmembrane helices and intracellular NH2 and COOH termini. SLC26A6 is expressed at highest levels in kidney and pancreas and, more specifically, in tubular cells in the kidney and in the apical surface of pancreatic ducts, suggesting it as a candidate for a yet-unknown cystic fibrosis transmembrane regulator (CFTR)-regulated protein responsible for luminal Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in pancreas (15).

The coordinated transport of ions and water across intact epithelium requires selective sorting of receptors, ion channels, and transporters to apical or basolateral cell surfaces. Recent studies demonstrated that PDZ (PSD-95/Disc-large/ZO-1) domains are protein-protein interaction domains that play an essential role in the assembly of multiprotein complexes ultimately involved in determining cell polarity, plasma membrane targeting, and regulation of membrane proteins (6). PDZ domains mediate interaction with the COOH terminus of proteins terminating in consensus PDZ binding sequences T/S-X-Phi (in which Phi  is a hydrophobic amino acid) (33). The CFTR has a highly conserved PDZ interaction motif (TRL), which has been shown to be required for binding to the closely related PDZ domain proteins Na+/H+ exchanger (NHE)3 regulatory factor (NHERF) and NHE3 kinase A regulatory protein (E3KARP) as well as to the CFTR-associated protein of 70 kDa (CAP70). This interaction has been implicated in the apical polarization and regulation of CFTR in epithelial cells (19, 21, 23, 25, 34, 37). Interestingly, the COOH terminus of SLC26A6 contains a consensus PDZ interaction motif identical to that of CFTR, raising the possibility of similar regulation pathways for the SLC26A6 protein. The characterization of the PDZ-SLC26A6 interaction is clearly warranted, because it could have important consequences for the regulation of SLC26A6 function and localization within cells.

We report the detection of two alternatively spliced variants of SLC26A6, named SLC26A6c and SLC26A6d, and their functional characterization. Transiently transfected proteins localized to plasma membrane and cytoplasm in nonpolarized cells, and functional expression in Xenopus oocytes generated Cl- and SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> transport. Furthermore, SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and Cl- transport were inhibited by DIDS and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. We show also that the COOH terminus of SLC26A6 interacts with the first and second PDZ domains of NHERF and E3KARP. Truncation of the COOH terminus abrogated the interaction but did not have an effect on transport function. These results demonstrate that SLC26A6 isoforms are novel AEs interacting with PDZ-domain proteins. SLC26A6 is a strong candidate for a CFTR-directed luminal Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in ductal pancreas.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Determination of alternative splicing. Alternative splicing was first detected when RT-PCRs to amplify the coding region of the SLC26A6 gene yielded several products. The PCR assays were done with 5'-ACC GAG GGA CAC ACA GGC ACT GCT-3' and 5'-ATG CAC CAG TTC CCT CCC TGT ACC-3' primers in 25-µl volumes with 2.5 µl of human kidney cDNA mix as template, 10 pmol of each primer, 1 × reaction buffer, each nucleotide at 0.2 mM, and 1 U of Advantage Polymerase Mix (Clontech, Palo Alto, CA) with the following conditions: 94°C for 3 min, 35 cycles of 94°C for 30 s and 68°C for 3 min, followed by 72°C for 10 min. The PCR products were separated on a 1% agarose-EtBr gel, cut, purified with a Qiagen gel extraction kit, and subcloned to pCR2.1-TOPO plasmid (Invitrogen). The specificity of the products was verified by sequencing with dye-terminator chemistry and an automated sequencer (ABI377; Applied Biosystems).

RNase protection analysis. The presence of the SLC26A6 isoforms was analyzed by the ribonuclease protection technique according to the Direct Protect kit (Ambion, Austin, TX) with two different RNA probes. A PCR fragment of 300 bp (bases 774-1075 in SLC26A6 cDNA; GenBank accession no. AF297659) to detect SLC26A6a and -A6c and another fragment of 363 bp (bases 2085-2316 +131 bp from the end of intron 16) to detect SLC26A6a and -A6d were amplified from human kidney cDNA (Clontech), purified, and subcloned to pCRII-TOPO (Invitrogen) to utilize the T7 and SP6 priming sites in the generation of the transcription templates. The orientation of the fragments was verified by sequencing. The transcription templates were amplified by PCR with the same forward primers as used in the previous PCR with T7 or SP6 oligo as an antisense primer. Antisense RNAs of 396 nt (probe 1) and 446 nt (probe 2) were transcribed with T7 or SP6 RNA polymerase by the Maxiscript SP6/T7 Kit (Ambion) in the presence of [alpha -32P]UTP and hybridized with 10 µg of total, 1 µg of poly A human kidney RNA, or 1 µg poly A human lung RNA. After an overnight hybridization, unpaired RNA was degraded by treatment with RNase cocktail at 37°C for 30 min followed by isopropanol precipitation. Protected RNA fragments were fractionated by 5% SDS-PAGE containing 6 M urea and visualized by autoradiography.

Tissue distribution of SLC26A6 isoforms. The tissue distribution of the splice variants was analyzed by PCR using Clontech's human multiple tissues cDNA panels I and II. The cDNA for Capan-1 was prepared as described previously (15). The isoform-specific regions were amplified with the following sense and antisense primers: SLC26A6a (351 bp) and -A6c (237 bp), 5'-GTG GGG CTG GGC CTG ATC CAC TTC-3'and 5'-GAC CCA TGC CAT AGG AGA TGC CTG-3'; SLC26A6d (890 bp), 5'-GAG ACT GGA GGT GGG AAA GGA GGT GAC AGC-3'and 5'-CTG CTG GGG AGC CAG ACA TGC TGCC-3'. PCR assays were performed in 25-µl volumes with 3 µl of each cDNA as template, 10 pmol of each primer, 1× cDNA reaction buffer (Clontech) reaction buffer, each nucleotide at 0.2 mM, and 1 U of Advantage Polymerase Mix (Clontech) with the following conditions: 94°C for 3 min, 35 cycles of 94°C for 30 s and 68°C for 1 min, followed by 72°C for 10 min. PCR products were run on a 2% agarose gel and stained with ethidium bromide to visualize expression patterns.

Specificity of antibodies. The specificity of the antibodies was analyzed by Western blotting with SLC26A6a- and SLC26A6c-transfected or untransfected COS-1 cells and by in vitro translation of the SLC26A6a protein. Specificity was shown also by competition with the antigenic peptides. Specific antibodies were raised in rabbits against the NH2-terminal amino acids, MDLRRRDYHMERPLLNQEHL, and COOH-terminal amino acids, TFALQHPRPVPDSPVSVTRL, corresponding to nucleotides 252-312 and 2405-2465 of the SLC26A6 cDNA sequence (AF279265), respectively. Peptide synthesis and antibody production were purchased from Sigma-Genosys (Cambridge, UK). Antibodies were purified from whole serum by affinity chromatography with the peptide coupled to N-hydroxysuccinimide-Sepharose 4B according to the manufacturer's instructions (Amersham Pharmacia Biotech). COS-1 cells were grown on 6-cm plates in Dulbecco's Eagle medium (GIBCO-BRL, Gaithersburg, MD) with 50 U/ml penicillin, 2 mM sodium pyruvate, 2 mM glutamine, and 5% fetal bovine serum at 37°C in 5% CO2 atmosphere. The cells were transiently transfected with Fugene6 (Roche Molecular Biochemicals) with either the SLC26A6a and -A6c clone (5 µg) or water as a control, following the manufacturer's instructions. Three days after the transfections, the cells were lysed in 500 µl of boiling Laemmli sample buffer (Pharmacia) with 5% beta -mercaptoethanol and separated by running on a 9% polyacrylamide gel before being transferred electrophoretically to a Hybond C-extra membrane (Amersham). Nonspecific binding sites were saturated by incubating the membranes in a solution of 5% nonfat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20. Proteins were detected by affinity-purified COOH-terminal anti-A6a antibodies (1 µg/ml) or NH2-terminal anti-A6a serum (1:200 dilution), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma) in PBS was used as a secondary antibody. NH2-terminal preimmune serum in the same concentration was used as control. For peptide competition analysis, affinity-purified COOH-terminal antibodies were incubated with ~100-fold excess of COOH-terminal A6a peptides at 37°C for 2 h before staining of the immunoblots or immunofluorescence. The protein bands were visualized by chemiluminescence reaction, and the signals were recorded by exposure to an X-ray film (Fuji). In vitro translation was performed according to the manufacturer's instructions (TNT T7 Quick Coupled Transcription/Translation System; Promega). In vitro translated SLC26A6a protein was detected by COOH-terminal antisera.

Transfection and immunofluorescence. Topologies of the SLC26A6 isoforms were analyzed in transfected permeabilized and nonpermeabilized COS-1 cells by immunofluorescence microscopy. The cDNAs of SLC26A6a, SLC26A6c, and SLC26A6d were amplified by PCR and subcloned to eukaryotic pcDNA3.1/V5/His-TOPO plasmid (Invitrogen). The same primers were used for template production as in the splicing analysis. For immunofluorescence staining, COS-1 cells plated on glass coverslips were transiently transfected with Fugene6 (Roche Molecular Biochemicals) with either the clones or water as a control, following the manufacturer's instructions. The cells were grown as described above. After 48 h, cells were fixed with 3% paraformaldehyde in PBS (0.14 M NaCl in 10 mM phosphate buffer, pH 7.4). After being fixed, the coverslips were washed two times with PBS and permeabilized with 0.1% Triton X-100 in PBS for 30 min and then continuing with blocking. If not permeabilized, the cells were washed after fixation and then blocked with 3% goat serum in PBS for 1 h. Affinity-purified antibodies were then added (1-5 µg/ml) in 1% goat serum in PBS and incubated for 1 h at room temperature. After three washes with 3% goat serum in PBS, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgGs (Sigma) were added and incubated for 1 h. Coverslips were then washed five times with PBS and mounted on glass slides with Immu-mount medium (Shandon, Pittsburgh, PA).

Xenopus oocyte injections and transport measurements. The cDNA sequences of SLC26A6a, SLC26A6c, SLC26d, SLC26A6adel, and SLC26A6cdel were amplified by PCR and subcloned to pCRII plasmids (Invitrogen). SLC26A6adel and SLC26A6cdel are COOH-terminal deletions of nine base pairs. A specific antisense primer (5'-CTA GAC CGA AAC AGG GCT GTC GGG GAC-3') missing the last nine bases but including a native stop codon was generated to delete the PDZ interaction motif (TRL) from SLC26A6adel and SLC26A6cdel constructs. The clones were verified by sequencing. For cRNA synthesis, plasmids were linearized by NotI digestion and transcribed in vitro with T7 RNA polymerase (Promega) and the resulting capped cRNAs were resuspended in water before use. Wild-type SLC26A3 cDNA was prepared as described previously (22). Mature Xenopus laevis females were purchased from the African Xenopus Facility C.C., (Noordhoek, South Africa). Stage V and VI oocytes from X. laevis were maintained at 17°C in modified Barth's solution [MBS; in mM: 88 NaCl, 1 KCl, 0.82 MgSO4, 0.4 CaCl2, 0.33 Ca(NO3)2, 2.4 NaHCO3, and 10 HEPES-Tris, pH 7.4, with 20 mg/l gentamicin sulfate]. Oocytes were injected with either 50 nl of water (control) or 3-7 ng of SLC26A6 cRNA isoforms with a Nanojet automatic injector (Drummond Scientific, Broomall, PA). Assay for transport of 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and 36Cl- uptake was performed on days 2 and 3 after injection. Briefly, 10 oocytes (per data point) were washed at room temperature for 1-2 min in solution A (in mM: 115 sodium gluconate, 2.5 potassium gluconate, 4 calcium gluconate, 10 HEPES-Tris pH 7.4) or solution B (in mM: 100 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, and 20 HEPES-Tris pH 7.5) and then placed into 100 µl of solution A containing 2.5 mM NaCl with 36Cl- or into 100 µl of solution B containing 0.1 mM K2SO4 with 10 µCi/ml 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> (New England Nuclear Radiochemicals) for 30-60 min at room temperature. The oocytes were washed four times with ice-cold solution A, lysed with 1% SDS, dissolved in biodegradable counting scintillant (BCS, Amersham), and counted by liquid scintillation spectrometry. Inhibition of the SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and Cl- uptake of SLC26A6 isoforms was performed by adding 1 mM DIDS, 10 mM NaHCO3, or 25 mM NaHCO3 to the uptake solution.

In vitro binding assays. The full-length cDNA sequences of SLC26A6a and SLC26A6adel in the pcDNA3.1/v5/his-TOPO plasmid were digested by HindIII (insert site) and EcoRV (vector site) and subcloned into pinPoint (Promega); these constructs were called Biotin-26A6-C-terminus and Biotin-26A6del-C-terminus. To express biotinylated fusion proteins, an overnight culture (pinPoint construct in Escherichia coli NM522) was diluted 1:100 in Luria broth (LB) plus 50 µM biotin and after 2.5 h 0.8 mM isopropylthiogalactoside (IPTG) was added. After another 4.5 h, bacteria were spun down and sonicated in 100 mM NaCl, 100 mM Na2HPO4, pH 7.4 in a Branson sonifier. The material was finally spun at 20,000 g for 20 min, and the supernatant was saved.

His-tagged fusion proteins of full-length NHERF (amino acids 1-367) and E3KARP (1-337) as well as their individual PDZ domains (NHERF-PDZ1: 1-153; NHERF-PDZ2: 159-346; E3KARP-PDZ1: 1-141; E3KARP-PDZ2: 130-311) and their COOH termini (NHERF: 266-368; E3KARP: 232-337) were expressed in pET30 (Novagen), affinity-purified under nondenaturing conditions with nickel-nitrilotriacetic acid (NTA) resin as suggested by the manufacturer (Qiagen), and finally eluted in 1 M imidazol, 150 mM NaCl, and 10 mM Na2HPO4, pH 8. The His-tagged fusion protein constructs of NHERF and E3KARP (~4 µg) were diluted in siliconized tubes in 1 ml of interaction buffer (200 mM NaCl, and 100 mM NaH2PO4, pH 7.5) to decrease the imidazol to <15 mM. One microliter (20 µl of 5% suspension supplied by the manufacturer) of magnetic Ni-NTA agarose beads (Qiagen) was added. After 1 h the beads were separated with a magnet, and the supernatant was removed. The beads were blocked for 10 min with 2% BSA in interaction buffer. Five hundred microliters of the cleared bacterial lysate of Biotin-SLC26A6-C-terminus and Biotin-26A6del-C-terminus and five hundred microliters of 4% BSA (in interaction buffer) were added, and the suspension was incubated for 4 h. The beads were washed four times with interaction buffer with the magnetic separator. Finally, the bound material was eluted in 50 µl of Laemmli sample buffer and separated on PAGE. After transfer, the nitrocellulose membranes were blocked with 3% BSA-Tris-buffered saline (TBS) and biotinylated proteins were detected with HRP-labeled streptavidin and an enhanced chemiluminescence (ECL) detection system.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Alternative splicing of SLC26A6. During the cloning of the full-length cDNA sequence of SLC26A6 (AF279265), several transcripts were repetitively obtained from RT-PCR amplifications and Northern blots, suggesting the presence of alternatively spliced forms of the SLC26A6 gene (15). Here we characterized two of them, named SLC26A6c ad SLC26A6d, in more detail. The clone nomenclature was chosen to portray the order of reporting these isoforms: our original SLC26A6 clone was labeled as SLC26A6a and its NH2-terminal variant, cloned by Waldegger et al. (36), as SLC26A6b. Sequence analysis of SLC26A6c and SLC26A6d revealed open reading frames of 2,100 bp and 2,016 bp, encoding 699- and 671-amino acid proteins, respectively. Comparison of the genomic structures and amino acid sequences of SLC26A6a and its splice variants is shown in Fig. 1, A and C. Gaps in SLC26A6c and SLC26A6d indicate the missing amino acids compared with SLC26A6a. The topology of SLC26A6a, SLC26A6c, and SLC26A6d analyzed by the PSIpred 2.0 method resulted in the prediction of 12-, 8-, and 12-transmembrane helices with intracytoplasmic NH2 and COOH termini, respectively (Fig. 1D). For comparison, an analysis by the hidden Markov model (TMHMM program) yielded 10-, 8-, and 10-transmembrane helices with intracellular NH2 and COOH termini for the three variants, respectively (Fig. 1D).


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Fig. 1.   A: genomic structure of SLC26A6 variants. The varying regions are shown in black, and the sizes of the deletion or insertion are marked above the altered exon or intron in SLC26A6c and SLC26A6d, respectively. The positions of RNase protection assay probes 1 (396 nt) and 2 (446 nt) are indicated. B: schematic presentation of the alternative first exons of the SLC26A6 gene. Translation initiation sites with Kozak sequence are marked by ATG. The last structure represents the alternative NH2-terminal variant of the SLC2A6 gene cloned by Waldegger et al. (36). C: comparison of the amino acid sequences of SLC26A6 variants. Transmembrane segments are shaded in gray. D: predicted transmembrane structure and hydropathy plot of SLC26A6 variants as determined by PSIpred2.0 and TMHMM programs.

Comparison of the nucleotide sequences of SLC26A6a and SLC26A6c revealed a 114-bp deletion within exon 6 and a 3-bp deletion in the beginning of the exon 17 produced by alternative usage of splice donor (GT) and acceptor sites, respectively (Fig. 1A). This results in a deletion of amino acids 243-281 and a glutamine residue (Q) at position 611 compared with SLC26A6a (AF279265). Deletion of the 38 residues results in the loss of two transmembrane domains (TMD 5 and 6) and predicts an additional large extracellular domain after TMD 4. A similar change was described previously in the analysis of alternative splicing of rat liver-specific organic anion transporter-1 (4). The extended extracellular loop might have effects on the properties of SLC26A6c.

The SLC26A6d sequence retains an unspliced intron, intron 16 (~800 bp), which includes several stop codons. This leads to an alternative COOH terminus of the protein compared with SLC26A6a, which may affect the regulation or function of SLC26A6d. Specifically, the COOH terminus of SLC26A6d lacks the PDZ interaction motif, which is included in SLC26A6a and SLC26A6c.

In addition to the splicing of the internal exons of SLC26A6a, the gene has several alternative first exons (Fig. 1B). The SLC26A6b cloned by Waldegger et al. (36) contains an additional NH2-terminal exon compared with our gene (Fig. 1B). This additional exon unites to our first exon 40 bp before our translation initiation site, leading to the translation of an additional 21 NH2-terminal amino acids. Both first exons have a putative translation initiation site with Kozak consensus sequence (Fig. 1B). However, in our SLC26A6 transcript, ~250 nucleotides precede the translation initiation site including four in-frame stop codons. Furthermore, two other putative first exons were predicted by the FGENES exon prediction program, and we verified them by sequencing the respective PCR fragments (data not shown). Both of these exons unite to our first exon and use the same translation initiation site we have proposed for SLC26A6a (Fig. 1B). Altogether, these findings propose that the SLC26A6 gene has different 5' ends and the SLC26A6b represents an additional NH2-terminal variant whose functional activity remains to be determined (36). The use of alternative first exons may result from the usage of distinct tissue-specific promoters, and further studies are required to find out the regulation and significance of the different SLC26A6 transcripts.

Similarly, two alternative 5' noncoding regions and translation sites with Kozak sequences were identified in the mouse slc26a6 gene (39). Inclusion of exon 1b in the longer slc26a6b transcripts results in a frameshift and a start codon within exon 2. The predicted slc26a6b protein is thus 23 amino acids shorter than slc26a6a. The start codons in both exon 1a and 2 are at good Kozak consensus sites. Slc26a6b is identical to the sequence of mouse Cl-/formate exchanger (CFEX) (13) and corresponds to our SLC26A6a, whereas the longer slc26a6a sequence corresponds to SLC26A6b reported by Waldegger et al. (36). The murine Slc26a6 and human SLC26A6 orthologs have similar genomic structures, but they share only 78% identity at the amino acid level (39).

To exclude PCR artifacts or incomplete splicing as explanations for the splice variants, the presence of the SLC26A6c and -A6d transcripts in human kidney and lung total RNA or mRNA were verified by ribonuclease protection assay (RPA). Specific antisense probes were designed for both splice variants to separate them from SLC26A6a (Fig. 1). RPA revealed one fragment of expected size for SLC26A6a (300 nt) with probe 1 (Fig. 2A). However, RPA did not detect the 200-bp fragment of SLC26A6c, because it is expressed very faintly if at all in human kidney, as shown by PCR analysis in Fig. 3A. However, similar results were observed with human lung mRNA (Fig. 2B), in which expression of SLC26A6c is higher than in kidney. Instead, two fragments of expected sizes for SLC26A6a and -A6d (225 and 356 nt, respectively) were protected with probe 2, confirming their expression in human kidney (Fig. 2A). SLC26A6d was expressed at approximately the same level as SLC26A6a.


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Fig. 2.   RNase protection analysis of SLC26A6 isoforms. A: total or poly A RNA from human kidney was hybridized with an antisense probe (probe 1, 370 nt) spanning exons 5-6 or with a probe (probe 2, 449 nt) spanning the end of intron 16 and exons 17-18 (see Fig. 1A). Unbound probe was degraded with RNases, and protected fragments were fractionated by 5% Tris-borate-EDTA (TBE)-PAGE containing 6 M urea and visualized by autoradiography. Ribonuclease protection assay (RPA) confirmed the presence of SLC26A6a and -A6d isoforms in the human kidney and SLC26A6a in the lung. Lanes 1 and 2, probe 1; lane 3, 10 µg of kidney total RNA with probe 1; lane 4, 1 µg of kidney mRNA with probe 1; lanes 5 and 6, probe 2; lane 7, 10 µg of kidney total RNA with probe 2; lane 8, 1 µg of kidney total RNA with probe 2. B: human lung mRNA was hybridized with probe 1. Lanes 1 and 2, probe; lane 3, lung mRNA.



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Fig. 3.   Expression of SLC26A6 isoforms. A: tissue distribution of the SLC26A6 variants was analyzed from Clontech's human multiple tissue cDNA (MTC) panels and Capan-1 cDNA by PCR. The SLC26A6a and -A6c isoforms were amplified in the same reaction. Sizes of the PCR products are shown. B: specificity of the antibodies was analyzed by immunoblotting. I: SLC26A6a- and SLC26A6c-transfected or water-transfected COS-1 cells were lysed and proteins were separated by SDS-PAGE before blotting to nitrocellulose membrane. Proteins were detected with affinity purified COOH-terminal anti-SLC26A6 antibodies, which detected the proteins of expected size of ~85 kDa. No bands were seen in water-transfected cells. Furthermore, the specificity of the bands was confirmed by peptide competition, which completely blocked immunostaining. II: in vitro translated SLC26A6a was detected by the affinity-purified COOH-terminal anti-A6 antibodies, whereas the rabbit normal IgG (used as control) remained negative. III: NH2-terminal anti-A6a serum detects the SLC26A6a protein. SLC26A6a- or water-transfected COS-1 cells were lysed and separated by SDS-PAGE before blotting to nitrocellulose membrane. NH2-terminal preserum was used as control for the same samples.

Tissue distribution of the SLC26A6a, SLC26A6c, and SLC26A6d variants were compared by PCR amplification of multiple tissue cDNA panels. Specific primers were designed to amplify both SLC26A6a and -A6c genes at the same reaction. SLC26A6a was detected in all tissues, whereas SLC26A6c showed a more restricted expression pattern (Fig. 3A). The expression of SLC26A6d gene was expressed in many tissues but mainly in the kidney and pancreas (Fig. 3A). All isoforms were expressed in the pancreatic ductal cell line Capan-1. These results suggest tissue-specific functions for different SLC26A6 variants. However, overlapping expression of SLC26A6 subtypes in some tissues suggests that expression might not be organ specific but also cell type specific.

Membrane topology of SLC26A6 protein isoforms. All of the SLC26 family members have been predicted to have 10-14 hydrophobic membrane domains and hydrophilic intracellular terminal tails; in some models, the COOH terminus has been predicted as extracellular. However, the exact topologies of the SLC26 anion exchangers are uncertain. To determine the location of the NH2 and COOH termini of the SLC26A6 variants relative to plasma membrane, anti-SLC26A6 antibodies specific to the NH2- and COOH-terminal tails were used in permeabilized and nonpermeabilized SLC26A6a, SLC26A6c-, and SLC26A6d-transfected cells. Transiently transfected COS-7 cells expressed all isoforms and demonstrated trafficking to plasma membrane as shown with permeabilized cells in Fig. 4, A-D and I, whereas untransfected cells remained negative (Fig. 4, K and L). Besides the membrane staining, staining of the intracellular organelles was observed as common for transiently transfected cells. This perinuclear staining most likely represents the overexpression of the newly synthesized proteins in Golgi and endoplasmic reticulum. However, no labeling was observed either with NH2-terminal or COOH-terminal antibodies in nonpermeabilized cells with SLC26A6a and -A6c isoforms, demonstrating that both the NH2 and COOH termini of the proteins are located intracellularly (Fig. 4, E-H). Similarly, the NH2 terminus of SLC26A6d was located intracellularly (Fig. 4J). The changed COOH terminus of SLC26A6d compared with SLC26A6a could not be localized, because we did not have a specific antibody against it. The specificity of the antibodies was confirmed by Western blotting, which showed that both NH2- and COOH-terminal antibodies bind specifically to the SLC26A6a and/or -A6c proteins in transfected cells compared with the untransfected cells (Fig. 3B). The molecular size of the protein bands (~85 kDa) corresponds well with the predicted sizes of the SLC26A6a and -A6c proteins (81 and 77 kDa, respectively). However, immunoblotting revealed some larger SLC26A6a bands, suggesting that the protein might undergo posttranslational modifications such as glycosylation. The specificity of the COOH-terminal antibodies was confirmed by a peptide competition assay, which completely blocked the staining of the protein bands (Fig. 3B). Similar results were also obtained with in vitro translated SLC26A6a protein. The membranes stained with preimmune sera remained negative (Fig. 3B). However, our results indicated that the NH2-terminal antibodies work much better for immunofluorescence than for immunoblotting. Altogether, our results support models with an even number of transmembrane helices, consistent with the predicted topology of SLC26A6a, SLC26A6c, and SLC26A6d proteins (Fig. 1D). Thus the topology of the SLC26A6 variants resembles the model suggested for SLC26A5, which is the closest homologue of SLC26A6 (15, 40). It follows from these results that the NH2 and COOH termini are possible sites of interaction with cytoplasmic proteins.


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Fig. 4.   Analysis of the membrane topology of SLC26A6 isoforms. Immunofluorescence images of transiently transfected COS-1 cells with SLC26A6a (A, B, E, F), -A6c (C, D, G, H), -A6d (I, J), or water (K, L). The cells were immunostained with NH2-terminal (A, C, E, G, I, J, K) and COOH-terminal (B, D, F, H, L) affinity-purified anti-SLC26A6 antibodies under permeabilized (A-D, I, K, L) and nonpermeabilized (E-H, J) conditions. Fluorescein isothiocyanate (FITC)-labeled rabbit IgG was used to locate the tails of the SLC26A6 isoforms. Images were captured with a ×63 objective.

SLC26A6 isoforms function as DIDS-sensitive AEs. The close homology of the SLC26A6 protein to the other members of the family suggests a related transport function, and a recent report (7) suggested AE activity for SLC26A6. To determine the functional activity of the different isoforms, we performed uptake measurements of SLC26A6a, SLC26A6c, and SLC26A6d cRNA-injected Xenopus oocytes with 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and 36Cl- alternatively as substrates. SLC26A3 cRNA was used as a positive control in transport experiments. Oocyte uptake experiments revealed ~10-fold induction of Cl- and ~17-fold induction of SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> transport compared with water controls (Fig. 5). Furthermore, the induced SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and Cl- uptakes were inhibited by the stilbene disulfonate DIDS (1 mM), an AE inhibitor, 10 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 5B), and 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (data not shown), suggesting that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> may possibly compete for the substrate binding site and thus may also be a substrate. Consistent with these findings, SLC26A6a was recently shown to function as a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in Xenopus oocytes (38). Similarly, Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger activity of mouse Slc26a6 has been demonstrated (10, 39). We also found that SLC26A6a induces oxalate transport (data not shown), as was also shown for the murine counterpart (10, 39). Altogether, these results demonstrate that the SLC26A6 isoforms function as AEs analogous to SLC26A3. The SLC26A3 protein is a major Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in the gut and transports at least SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, oxalate, and OH- as well (31, 18, 22), whereas SLC26A4 transports at least I-, Cl-, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, OH-, and formate but not SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> (28). Functional activity of SLC26A6a, SLC26A6c, and SLC26A6d in Xenopus oocytes also demonstrates trafficking of the proteins to the cell membrane.


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Fig. 5.   Functional expression of SLC26A6 isoforms in Xenopus oocytes. Oocytes were injected with water or SLC26A6 (A6a), SLC26A6c (A6c), SLC26A6d (A6d), SLC26A6adel (A6adel), SLC26A6cdel (A6cdel), or SLC26A3 cRNA. Transport of 36Cl- (A) and 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> (B) uptake was performed (see EXPERIMENTAL PROCEDURES) as well as the inhibition of the SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> and Cl- uptake by 1 mM DIDS and 10 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Data are shown as means ± SE for 7-10 oocytes per condition and are representative of 3 similar experiments.

COOH terminus of SLC26A6 and s1 contains a functional PDZ domain. The ultimate COOH terminus of SLC26A6a and SLC26A6c contains a PDZ-interaction motif identical to that of CFTR (TRL), prompting us to test its putative binding to different PDZ domains of the E3KARP and NHERF proteins in vitro. The intact COOH terminus of SLC26A6a and -A6c, but not the SLC26A6adel mutation missing the PDZ interaction motif, bound to full-length NHERF and E3KARP. This result demonstrates the functionality and specificity of the COOH-terminal TRL motif of SLC26A6 and SLC26A6c for interaction with NHERF and E3KARP. To test whether this interaction indeed occurred through the PDZ domains of these proteins, the PDZ domains and the COOH terminus of NHERF and E3KARP were individually tested for binding. SLC26A6 was found to bind to both PDZ domains but not to the COOH terminus of NHERF and E3KARP (Fig. 6). Furthermore, the truncation of the COOH terminus of SLC26A6a and SLC26A6c did not affect Cl- transport function (Fig. 5).


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Fig. 6.   Interaction of SLC26A6a with the PDZ domains of Na+/H+ exchanger (NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor (NHERF). Hexahistidine-tagged constructs of NHERF and E3KARP, their individual PDZ domains, and their COOH termini were expressed in Escherichia coli and immobilized on nickel-agarose. Their ability to precipitate a biotinylated construct of the COOH terminus of SLC26A6 (+) or a mutated construct lacking the potential PDZ interaction motif TRL (-) was tested. Copurified material was separated on PAGE, and biotinylated proteins were detected with streptavidin-horseradish peroxidase (HRP) conjugate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The SLC26 family of proteins, earlier designated as SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> transporters, has been redefined as AEs with additional functionalities (the motor activity of SLC26A5) (3, 5, 8, 9, 12, 15, 16, 18, 22, 24, 27-29, 35, 41). Many observations feed the growing interest in this family, including the involvement of the three first members, SLC26A2-A4, in phenotypically different recessive diseases, the existence of several new tissue-specific members, and the concept that transport proteins may be organized in membrane microdomains through specific interacting proteins and the cytoskeleton. In this report, we have verified two key functions for a recently cloned member of this family. SLC26A6a and its two novel splice variants, SLC26A6c and SLC26A6d, function as novel DIDS-sensitive AEs. The length of the SLC26A6d transcript corresponds well with the previously observed size (~3.7 kb) of the bigger transcript in a Northern blot (15). We have also demonstrated that SLC26A6a interacts with the PDZ domain proteins NHERF (EBP50) and E3KARP in vitro. Because the CFTR-dependent activation of epithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is suggested to be mediated by PDZ interactions, our results support a function for SLC26A6 as a novel CFTR-directed Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, possibly responsible for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion in stimulated pancreatic ductal cells. SLC26A6a does not have a motor function similar to SLC26A5 (24).

SLC26A6 protein was previously located immunohistochemically to the apical and basolateral surfaces of tubular walls of human kidney and the apical surfaces of ductal pancreas (15). Moreover, all SLC26A6 isoforms are expressed in a pancreatic ductal cell line, Capan-1. Demonstration of Cl-, SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and oxalate transport activities further supports SLC26A6 as a novel Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger abundantly expressed in pancreas and kidney. This is also supported by the high structural homology of SLC26A6 to known family members (SLC26A3-A4), which both transport Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (18, 26, 32). Finally, a recent study demonstrated that SLC26A6a indeed functions as a DIDS-sensitive Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger when expressed in Xenopus oocytes (38). This same study identified SLC26A6 as an apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in the mouse gastrointestinal tract (38).

The expression pattern and substrate specificity of the mouse slc26a6 suggest that it mediates the exchange of different anions in a number of tissues, supporting our observations. Recently, a putative mouse ortholog of human SLC26A6a, CFEX, (Cl-/formate exchanger), was localized to the brush border membrane of renal proximal tubule cells and was demonstrated to mediate Cl-/formate exchange when expressed in Xenopus oocytes (13). Further studies on different anions demonstrated that mouse slc26a6 has affinity for oxalate, SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> in addition to Cl- and formate (10). Mouse Slc26a6 can also function in multiple exchange modes involving different pairs of these anions, sharing the ability to mediate Cl-/base exchange with slc26a3 and slc26a4 (18, 32, 10, 39). Interestingly, the mouse slc26a6-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or Cl-/oxalate exchange induces simultaneous membrane hyperpolarization, suggesting that it is an electrogenic transporter (10, 39). More detailed physiological characterization and expression studies of different human SLC26A6 variants will provide further information for understanding their physiological role in different tissues.

Ductal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion and Cl- absorption are tightly coupled in pancreas and mediated in part by a luminal Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger regulated by CFTR (1, 14). The regulatory role of CFTR is supported by CFTR mutations that retain channel Cl- activity but lead to marked reduction in Cl-- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-dependent fluid secretion in the pancreatic juice of cystic fibrosis patients (11). Recently, in the mouse pancreatic duct, CFTR was found necessary for the cAMP inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> salvage, which is mediated by NHE3 (SLC9A3) and a yet unidentified Na+-dependent system. This regulatory interaction is mediated by binding of CFTR to PDZ1 and NHE3 to PDZ2 of NHERF (also known as EBP50) (1). Moreover, CFTR was shown to upregulate the expression of the NHE3, SLC26A3, and SLC26A6 mRNAs (7). Our results, showing the interaction of SLC26A6 with PDZ1-2 domains of NHERF and E3KARP, suggest a similar regulatory mechanism for CFTR-directed luminal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion involving a CFTR-SLC26A6-NHERF complex. On pancreatic cell stimulation, a complex of CFTR, NHERF (or E3KARP), and SLC26A6 might be assembled (or a preexisting complex might be activated or modified) to stimulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, while the CFTR-NHERF-NHE3 complex inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> salvage.

This coarse model will obviously need further refinement. Despite a number of studies, the functional role of the PDZ interaction motif of CFTR remains somewhat unclear. Sophisticated kinetic analyses have revealed that the COOH terminus of CFTR binds to both PDZ domains of NHERF (30, 34), but the CFTR-PDZ1 complex forms much faster than CFTR-PDZ2 complex, whereas the CFTR-PDZ2 remains more stable than the CFTR-PDZ1 complex (25). PDZ interaction appears to be important, but not sufficient, for targeting of CFTR to or retention of CFTR in the apical membrane in polarized cells (20). Recently, it was demonstrated that the binding of NHERF or another PDZ domain protein, CAP70, to CFTR has a direct potentiating effect on CFTR channel activity (25, 37). With regard to SLC26A6a and SLC26A6c (which carry the PDZ interaction motif) and SLC26A6d (which does not have the PDZ interaction motif), further studies will need to address the functional significance of the PDZ domain interaction motif for plasma membrane targeting and modulation of transport activity in polarized cells. Also, the exact composition of a putative multiprotein complex of NHERF or E3KARP, SLC26A6, and possibly CFTR or other proteins remains to be determined.


    ACKNOWLEDGEMENTS

We thank Ranja Eklund for skillful assistance in laboratory work.


    FOOTNOTES

This study was supported by Academy of Finland; Sigrid Juselius Foundation; Foundation for Pediatric Research, Ulla Hjelt Fund; Helsinki University Central Hospital research funds; the National Health and Medical Research Council of Australia (D. Markovich); Oskar Öflund Foundation; Research and Science Foundation of Farmos; Emil Aaltonen Foundation; The Kidney Foundation; Finska Läkaresällskapet; and Deutsche Forschungsgemeinschaft Grant La1066/2-1 (G. Lamprecht). J. Kere is a member of Biocentrum Helsinki.

Address for reprint requests and other correspondence: H. Lohi or J. Kere, Dept. of Medical Genetics, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), Univ. of Helsinki, 00014 Helsinki, Finland (E-mail: hannes.lohi{at}helsinki.fi or juha.kere{at}helsinki.fi).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 20, 2002;10.1152/ajpcell.00270.2002

Received 11 June 2002; accepted in final form 30 October 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 284(3):C769-C779
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