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
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ABSTRACT |
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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
was inhibited by DIDS and HCO
alternative splicing; PDZ domain; SLC26
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INTRODUCTION |
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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
,
HCO
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
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- (in which
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
transport were inhibited by DIDS and
HCO
/HCO
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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
[-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%
-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
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
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. ![]() |
RESULTS |
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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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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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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 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
uptakes were inhibited by the stilbene disulfonate DIDS (1 mM), an AE
inhibitor, 10 mM HCO
/HCO
/HCO
/HCO
as well (31, 18, 22), whereas SLC26A4
transports at least I
, Cl
,
HCO
, and formate but not
SO
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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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DISCUSSION |
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The SLC26 family of proteins, earlier designated as
SO/HCO
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
/HCO
and HCO
/HCO
/HCO
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
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
/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 absorption
are tightly coupled in pancreas and mediated in part by a luminal
Cl
/HCO
activity but lead to
marked reduction in Cl
- and
HCO
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.
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ACKNOWLEDGEMENTS |
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We thank Ranja Eklund for skillful assistance in laboratory work.
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FOOTNOTES |
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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.
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