We report the molecular and
functional characterization of murine Slc26a6, the putative apical
chloride-formate exchanger of the proximal tubule. The Slc26a6
transcript is expressed in several tissues, including kidney.
Alternative splicing of the second exon generates two distinct
isoforms, denoted Slc26a6a and Slc26a6b, which differ in the inclusion
of a 23-residue NH2-terminal extension. Functional
comparison with murine Slc26a1, the basolateral oxalate exchanger of
the proximal tubule, reveals a number of intriguing differences.
Whereas Slc26a6 is capable of Cl
, SO
,
formate, and oxalate uptake when expressed in Xenopus
laevis oocytes, Slc26a1 transports only SO and oxalate. Measurement of intracellular pH
during the removal of extracellular Cl in the presence
and absence of HCO
indicates that Slc26a6 functions
as both a Cl/HCO and a
Cl/OH exchanger; simultaneous membrane
hyperpolarization during these experimental maneuvers reveals that
HCO and OH transport mediated by
Slc26a6 is electrogenic. Cis-inhibition and efflux
experiments indicate that Slc26a6 can mediate the exchange of both
Cl and SOwith a number of substrates, including formate and oxalate. In contrast, SO and
oxalate transport by Slc26a1 are mutually cis-inhibited but activated significantly by extracellular halides, lactate, and formate.
The data indicate that Slc26a6 encodes an apical
Cl/formate/oxalate and Cl/base exchanger
and reveal significant mechanistic differences between apical and
basolateral oxalate exchangers of the proximal tubule.
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INTRODUCTION |
ANION EXCHANGE AT THE
PLASMA membrane is primarily mediated by the products of two
structurally distinct gene families; the anion exchanger (AE) genes,
which form a subset of the bicarbonate transporter, SLC4
superfamily (41, 52), and the SLC26 or
"sulfate permease" gene family (10, 33). The mammalian
SLC26 gene family has emerged over the last seven years
through a combination of expression cloning (4),
subtractive cDNA cloning (61), and positional
characterization of human disease genes (10). Evolving physiological roles for specific family members include transepithelial salt transport (10, 45), thyroidal iodide transport
(46), development and function of the inner ear (10,
61), sulfation of extracellular matrix (43), and
the renal excretion of both bicarbonate (42) and oxalate
(17). The various substrates transported by the SLC26
anion exchangers include sulfate (SO), chloride
(Cl), iodide (I), formate, oxalate,
hydroxyl ion (OH), and bicarbonate
(HCO) (4, 17, 36, 43, 45, 49).
The SLC26 gene family is highly conserved across evolution, with
identifiable homologues in bacteria, fungi, yeast, plants, and animals
(10). The Drosophila genome contains at least
nine family members, and the existence of new mammalian paralogs has been suspected for some time (10). The cloning of several
of these new genes has been recently reported (19, 30, 31, 53,
54), and the family appears to encompass 10 members and 1 pseudogene (Mount DB, unpublished observations). Physiological interest in the characterization of novel family members was stimulated in particular by the observation that SLC26A4 can function as a
Cl/formate exchanger (45), because
Cl/formate exchange is thought to play a pivotal role in
the transepithelial transport of NaCl by the renal proximal tubule
(56, 57). However, Slc26a4 is not expressed in the
proximal tubule but is instead found at the apical membrane of
-intercalated cells, where it appears to play a role in renal
bicarbonate excretion (42). The sixth member of the gene
family, SLC26A6, was recently cloned in both humans
(SLC26A6) (30, 54) and mice
(Slc26a6, also known as CFEX) (19). Functional
characterization of Slc26a6 indicates that it can mediate both
Cl/formate and Cl/Cl exchange
(19), whereas the human ortholog was nonfunctional (54). The immunolocalization of SLC26A6 and Slc26a6
indicates expression at the apical membrane of epithelial cells
(19, 30) and suggests that this gene encodes the proximal
tubule Cl/formate exchanger. Important unresolved issues
include whether SLC26A6 also mediates Cl/oxalate exchange
and Cl/base exchange (Cl/OH
and/or Cl/HCO exchange). Apical
oxalate transport by a DIDS-sensitive anion exchanger is thus thought
to play an important role in oxalate secretion by the proximal tubule,
in concert with basolateral oxalate transport mediated by Sat-1
(SLC26A1/Slc26a1). There is also a significant body of evidence
suggesting that apical Cl/base exchange functions in
transepithelial NaCl absorption by the proximal tubule
(24). Moreover, the ability of SLC26 proteins to function
as Cl/base exchangers identifies Slc26a6 as a candidate
for both the apical Cl/base exchanger of the proximal
tubule and the apical CFTR-dependent bicarbonate transporter(s) in the
lung (26), submandibular gland (27), and
exocrine pancreas (7, 27). We report the initial exploration of these issues, in addition to a functional comparison of
the murine Slc26a1 and Slc26a6 anion exchangers.
| |
METHODS |
Molecular characterization of Slc26a6 and Slc26a1.
Human SLC26A6 exons were initially identified in the draft
sequences of the BAC clone RP11-148G20 and the PAC clone
RP4-751E10, using tblastn searches of the HTGS database
with the SLC26A1-4 proteins. A blastn search of the
mouse expressed sequence tag (EST) database using the extracted human
exon contig yielded a Sugano mouse IMAGE clone (2076921), with 5'- and
3'-EST entries that exhibited modest homology to the NH2
and COOH termini of known SLC26 proteins. This full-length cDNA was
obtained from Research Genetics (Birmingham, AL), sequenced on both
strands using fluorescent dye terminator chemistry (Applied
Biosystems), and submitted to GenBank (AF248494, 3/23/00). A
blastn search of the Celera mouse genomic database yielded a
500-kb contig containing the 11-kb Slc26a6 gene, and a
subsequent blastn search of the mouse EST database using a
1.7-kb region between the start of the 2076921 EST and the
3'-untranslated region (UTR) of the upstream gene yielded three RIKEN
5'-ESTs that overlap with 2076921 but have a different 5'-end. This
alternative 5'-end was cloned by RT-PCR from mouse intestine total RNA,
using a sense primer in exon 1a (5'-TACACGAGTTACCCTCTGAGG-3') and an
antisense primer from within exon 4 (5'-TACAGACCAAACATAGGAGGC-3'), as
described (37). The two amplified PCR fragments obtained
(see Fig. 2) were subcloned into pCR2.1 (Invitrogen) and sequenced.
Finally, for the purpose of functional comparison to Slc26a6, we
identified and sequenced a full-length mouse Slc26a1 EST (IMAGE clone 1450460).
The analysis of nucleotide and amino acid sequence utilized
Vector NTI 6.0 (Informax), supplemented by GRAIL
(http://compbio.ornl.gov/Grail-1.3/), Phosphobase
(http://www.cbs. dtu.dk/databases/PhosphoBase/), MattInspector (http://transfac. gbf.de/cgi-bin/matSearch/matsearch.pl), TESS (http://www. cbil.upenn.edu/cgi-bin/tess/tess33?RQ=SEA-FR-Query), and
Prosite (http://www.expasy.ch/prosite/). Genomic localization of
Slc26a1 and Slc26a6 exploited Celera genomic
contigs encompassing the two genes, the UniSTS website (NCBI), and the
Mouse Genome Database (MGD;
http://www. informatics.jax.org/mgihome/).
Northern blot analysis.
RNA was extracted from C57BL/6J mice using guanidine isothiocyanate and
cesium chloride. Total RNA (10 µg/lane) was size-fractionated by
electrophoresis (5% formaldehyde, 1% agarose), transferred to a nylon
membrane (Stratagene), and probed sequentially with 32P-labeled randomly primed probes corresponding to
full-length GAPDH and a 3'-probe from Slc26a6 (nucleotides
2339-2673 of Slc26a6b). Hybridization was performed overnight at
42°C in Express-Hyb solution (Clontech), and membranes were washed
twice for 10 min at room temperature in 2× SSCP/0.1% SDS and twice
for 1 h at 65°C in 0.1× SSCP/0.1% SDS.
Expression of Slc26a1 and Slc26a6 in Xenopus laevis oocytes.
The entire inserts of the Slc26a6b and Slc26a1 cDNAs were transferred
to the pGEMHE X. laevis expression vector (29)
using EcoRI and XbaI. The Slc26a6b and Slc26a1
expression constructs were linearized using NheI and
NotI, respectively, and cRNA was transcribed in vitro using
T7 RNA polymerase and mMESSAGE mMACHINE kits (Ambion). Defolliculated
oocytes were injected with 25-50 nl of water or a solution
containing cRNA at a concentration of 0.5 µg/µl (12.5-25
ng/oocyte), using a Nanoliter-2000 injector (WPI Instruments, Sarasota,
FL). Oocytes were incubated at 17°C in 50% Leibovitz's L-15 media
supplemented with Pen-Strep (1,000 U/ml) and glutamine for 2-3
days before uptake assays.
For sulfate uptakes, oocytes were preincubated for 20 min in
chloride-free uptake medium [100 mM
N-methyl-D-glucamine (NMDG)-gluconate, 2 mM
potassium gluconate, 1 mM calcium gluconate, 1 mM magnesium gluconate,
10 mM HEPES-Tris, pH 6.0 or pH 7.5, as indicated], followed by a
60-min uptake in the same medium with 1 mM
K235SO4. The cells were then washed
three times in uptake buffer with 5 mM nonradioactive
K2SO4 to remove tracer activity in the
extracellular fluid. The oocytes were dissolved individually in 10%
SDS, and tracer activity was determined by scintillation counting.
Cl, formate, and oxalate uptakes were assayed using the
same Cl-free uptake solutions, substituting 8.3 mM
36Cl, 500 µM [14C]oxalate, or
50 µM [14C]formate for labeled sulfate. For sulfate
exchange (see Fig. 5C) and cis-inhibition (Fig.
5A), the concentration of NMDG-gluconate in the uptake
solution was adjusted to maintain isotonic osmolality, which was
experimentally confirmed using a Fiske 110 osmometer. Cis-inhibition experiments used the Na+ salts of
the relevant anions. All radioisotopes were from New England Nuclear
(Boston, MA).
The uptake experiments all included 12-18 oocytes in each
experimental group, statistical significance was defined as two-tailed (P < 0.05), and results are reported as means ± SE.
Oocyte electrophysiology.
Oocytes were studied 3-11 days postinjection. The
CO2/HCO-free ND96 contained 96 mM NaCl,
2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM
HEPES (pH 7.5 and 195-200 mosmol/kgH2O) and bubbled
with 100% O2 during experiments. For CO2/HCO-equilibrated solutions, 33 mM
NaHCO3 replaced 33 mM NaCl. In 0-Na+ solutions,
choline replaced Na+. In 0-Cl solutions,
gluconate replaced Cl. All solutions were titrated to pH
7.5 and continuously bubbled with CO2-balanced
O2 to maintain PCO2 and pH.
Ion-selective microelectrodes were prepared and calibrated, and
experiments were performed as previously described for NBC
(40) and NDAE1 (41). All pH electrodes had
slopes of at least 
56 mV/decade concentration change. Where statistical tests were indicated, a two-tailed Student's
t-test assuming unequal variances was used. P
values <0.05 were considered statistically significant.
| |
RESULTS |
Molecular biology.
Sequence comparison indicates that the murine Slc26a6 and human SLC26A6
orthologs share only 78% identity at the amino acid level, much lower
than the median of 86% for human and mouse orthologous pairs
(32). However, the murine and human genes are clearly orthologous, such that both are flanked at the 5'-ends by the FMI-1/MEGF-2 gene and at the 3'-end by the UQCRC1
and ColA7 genes (13, 28). The gene and sequence
tagged site content of these contigs confirm that the human
SLC26A6 gene is on chromosome 3p21 (30) and the
murine gene is in the syntenic segment of chromosome 9, at ~61
cM. In particular, the murine ColA7 gene is
physically linked ~40 kb 3' of Slc26a6 and is known to be
localized on mouse chromosome 9 at 61.0 cM (UniSTS marker 859381)
(28).
The Slc26a6 and SLC26A6 genes share a similar
organization, encompassing 21 coding exons and ~10 kb of genomic
DNA. Both genes include an alternative 5' noncoding exon (exon 1b, 945 nucleotides 5' of coding exon 2 in Slc26a6). Nonquantitative RT-PCR
using primers in exons 1a and 4 (see Fig. 2A) suggests that
the isoform in which exon 1b has been spliced out, denoted Slc26a6a, is
expressed at a lower level than Slc26a6b; our functional experiments
utilized a Slc26a6b construct. The identity of the two RT-PCR fragments (see Fig. 2A) was verified by subcloning and sequencing of
the amplified bands. The intron-exon boundaries in Slc26a6
were determined by comparison of the various Slc26a6 cDNAs to the
murine Celera contig, and all of the donor and acceptor sites were
found to conform to consensus splice sites (39) (Table
1). The inclusion of exon 1b in the
longer Slc26a6b transcript results in a frame shift and a start codon
within exon 2. The predicted Slc26a6b protein is thus 23 amino acids
shorter than Slc26a6a (Fig. 1). The start
codons in exon 1a and exon 2 are both predicted Kozak sites
(21), with purines at position 3 and G at position +4. However, it is conceivable that the Kozak site in exon 2 is in fact
preferred for translation initiation in both the Slc26a6a and Slc26a6b
transcripts; formal proof that the start codon in exon 1a is utilized
will require the generation of an antibody to the putative
NH2-terminal extension of the Slc26a6a protein. Slc26a6b is
essentially identical to the sequence of mouse "CFEX" (19), except for codons 2 (glutamate in Slc26a6b, glycine
in CFEX), 65 (valine in Slc26a6b, indeterminate in CFEX), and 549 (proline in Slc26a6b, arginine in CFEX). Exon 1b is conserved in the
human genomic sequence; the cDNA reported by Waldegger et al.
(54) corresponds to SLC26A6a, whereas that reported by Lohi et al. (30) corresponds to SLC26A6b. The functional
significance of the alternative NH2 terminus in the
Slc26a6a isoform is not yet known; however, note is made of the
conservation of the sequence, TQALLS, in mice, humans, and pigs
(Fig. 1 and data not shown, porcine SLC26A6a). The murine Slc26a6b
isoform lacking the NH2-terminal extension is functional
(see Figs. 4-7); hence, this sequence is not required for
transport activity.
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Fig. 1.
Sequence analysis of the Slc26a6 and Slc26a1 proteins.
A: an alignment of the first 100 amino acids of the murine
Slc26a6 and human SLC26A6 proteins. The unique NH2-terminal
extensions predicted in the longer Slc26a6a/SLC26A6a proteins are in
bold text and boxed in green; the Slc26a6b protein begins at the
methionine after this extension. Predicted PKC phosphorylation sites
are in yellow. B: predicted sequence of the murine Slc26a1
protein. N-glycosylation sites are in boxed green, PKC sites
in yellow, PKA sites in red, combined PKC/PKA sites in red-outlined
yellow, and tyrosine kinase sites in orange. Potential transmembrane
domains are underlined.
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Northern blot analysis indicates that the Slc26a6 gene is
widely expressed, with a 3.0-kb transcript detected in intestine, kidney, testis, brain, muscle, heart, and stomach (Fig.
2B); hybridization levels for
GAPDH were approximately equal (not shown). The widespread expression
of Slc26a6 is consistent with the presence of a CpG island overlapping
exon 1a in the Celera genomic contig (Fig. 3), conserved in the human gene (data not
shown). Although the transcriptional start site has not been mapped for
either species, the most 5' Slc26a6a ESTs begin ~100 bp 5' of the
start codon in exon 1a. The genomic DNA flanking in exon 1a suggests a
TATA-less promoter, rich in predicted Sp1 sites (Fig. 3).
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Fig. 2.
Expression of Slc26a6 transcripts. A: RT-PCR of mouse
tissues, using a sense primer in exon 1a and an antisense primer in
exon 4. Reaction products were run on a 6% acrylamide gel; controls
include both water and an RT-negative () sample. The Slc26a6a
transcript yields a 300-bp band due to alternative splicing of the 5'
exon 1b, whereas the Slc26a6b transcript yields a 438-bp band due to
retention of this exon. B: mouse multiple-tissue Northern
blot, probed with a 3' probe specific for Slc26a6 (4-day exposure).
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Fig. 3.
Sequence of the proximal promoter from the Slc26a6 gene. Genomic
sequence surrounding exon 1a is displayed. This data was generated
through the use of the Celera Discovery System and Celera's associated
databases. Coding sequence from the 3'-end of exon 1a is underlined in
bold, beginning with the predicted "ATG" start codon. The predicted
CpG island is indicated by bold brackets. Potential binding sites for
transcription factors, predicted using TESS and Mattinspector, are
boxed and labeled.
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For the purpose of functional comparison to Slc26a6, we also
characterized mouse Slc26a1 (Sat-1), identified on a full-length EST
cDNA (Fig. 1B). The predicted Slc26a1 protein is 91 and 76% identical to the rat and human SLC26A1 proteins, respectively. As is the case with Slc26a6/SLC26A6, the mouse
and human Slc26a1/SLC26A1 genes are clearly
orthologous. Large genomic contigs containing the mouse (Celera) and
human (GenBank NT_006111) genes reveal a conserved organization, such
that both are flanked at the 5'-end by the FGFRL-1
(59), GAK (18), and
DAGK4 (9) genes and at the 3'-end by the
L-iduronidase gene. The murine
FGFRL-1 and L-iduronidase genes have
both been localized on mouse chromosome 5 at ~57 cM
(59), syntenic with the region of human chromosome 4p16
containing SLC26A1, GAK (18), and
DAGK4 (9). The genomic organization of the two
SLC26A1/Slc26a1 genes is also conserved, although
analysis of a number of 5' Slc26a1 ESTs reveals the existence of two 5'
noncoding exons in the murine gene (Table
2). The more 3' of these two noncoding
exons, denoted exon 1b, is excluded from a number of ESTs, indicating
that it is alternatively spliced. Of note, the relative position of the
junction between the two coding exons of
SLC26A1/Slc26a1 and SLC26A2 (DTDST),
which together form a separate branch of the gene family, is conserved
in the respective mouse and human genes.
Anion transport in X. laevis oocytes.
We have characterized the physiological properties of mouse Slc26a6b
and Slc26a1 in X. laevis oocytes using similar protocols to
those published for SLC26A1 (4, 25, 43), SLC26A2
(43), SLC26A3 (36), and SLC26A4 (45,
46).
Figure 4 illustrates the uptake of
35SO and 36Cl
by oocytes injected with Slc26a1 and Slc26a6, as a function of extracellular pH and extracellular Cl or
SO. As shown in Fig. 4A, Slc26a1 mediates Cl- and Na+-independent sulfate
transport (148 ± 9 and 237 ± 37 pmol · oocyte1 · h1 at pH
7.4 and 6.0, respectively, vs. 2.0 ± 0.2 and 5.8 ± 0.9 pmol · oocyte1 · h1 in
water-injected controls), as does Slc26a6 (605 ± 40 and 625 ± 53 pmol · oocyte1 · h1
at pH 7.4 and 6.0, respectively). The difference in
35SO uptake between pH 7.4 and 6.0 is
significant for Slc26a1 (P < 0.02) but not for
Slc26a6. Both transporters are more sensitive to 1 mM DIDS at the lower
pH (301 ± 24 pmol · oocyte1 · h1 at pH
7.4 vs. 101 ± 10 pmol · oocyte1 · h1 at pH
6.0 for Slc26a6 in the presence of DIDS). In contrast, Cl
uptake in Slc26a6/CFEX-expressing oocytes was reported by Knauf et al.
(19) to be highly sensitive to 100 µM DIDS, with no
significant difference between pH 7.4 and 6.5; this higher sensitivity
may have masked an effect of extracellular pH. It is also likely that DIDS sensitivities of Slc26a6 and other members of the family are
highly dependent on both the concentration and the identity of the
transported anion (16), although we note that SLC26A3 and
SLC26A4 are also only modestly sensitive to DIDS (1, 46).
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Fig. 4.
Functional characterization of Slc26a1 and Slc26a6:
Cl and sulfate uptakes. A: uptake of
35SO uptake in Slc26a6- and
Slc26a1-injected oocytes
(pmol · oocyte1 · h1), with
extracellular pH of both 7.4 (open bars) and 6.0 (filled bars) and in
the presence and absence of 1 mM DIDS. B: Cl
(36Cl) uptake by Slc26a1- and
Slc26a6-injected oocytes, with extracellular pH of both 7.4 (open bars)
and 6.0 (filled bars). The second group of Slc26a1 oocytes (Slc26a1,
SO4) was incubated in medium containing 25 mM SO
during the uptake in an attempt to stimulate Cl uptake
(see text). C: differential effect of the presence of
extracellular Cl on 35SO
uptake by Slc26a1 and Slc26a6. Oocytes were incubated in
35SO uptake medium for 1 h in the
absence (open bars) or presence (gray bars) of 25 mM extracellular
Cl. Whereas 35SO uptake
by Slc26a6 is cis-inhibited by extracellular
Cl, 35SO uptake in
Slc26a1-injected oocytes is increased in the presence of extracellular
Cl. D: effect of extracellular pH on
35SO uptake in the presence of 25 mM
extracellular Cl, at pH 7.4 (open bars) and 6.0 (filled
bars). *Significantly different (P < 0.01) from
water-injected control oocytes.
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Oocytes expressing Slc26a6 transported 36Cl
(Fig. 4B), although, again, there was no consistent
difference between pH 7.4 and 6.0 for Slc26a6-injected groups
(4,345 ± 243 and 4,193 ± 109 pmol · oocyte1 · h1 at pH
7.4 and 6.0, respectively, vs. 106 ± 12 and 99 ± 16 pmol · oocyte1 · h1 in
water-injected controls). In contrast to Slc26a6, Slc26a1-injected oocytes did not take up 36Cl; this is in
agreement with transport studies using basolateral membrane vesicles
from renal cortex, which indicate that the basolateral SO/HCO exchanger in these
preparations does not transport Cl (23). We
considered that Slc26a1 might require the presence of extracellular
SO to transport Cl; however, Slc26a1
mediates minimal Cl uptake in either the presence or
the absence of 25 mM SO (Fig. 4B).
The absolute rates of 35SO uptake for
Slc26a1-injected oocytes shown in Fig. 4A are reproducibly lower than those of Slc26a6-injected oocytes. However,
SO uptake rates for Slc26a1 are much higher in the
presence of extracellular Cl, closer to the values for
Slc26a6 in the absence of Cl (Fig. 4C). In
contrast, 35SO uptake by
Slc26a6-injected cells is strongly cis-inhibited by
extracellular Cl (Fig. 4C), consistent with
the ability of this exchanger to mediate 36Cl
uptake (Fig. 4B). The increase in
35SO transport by Slc26a1 at an
extracellular pH of 6.0 is greater in the presence (Fig.
4D) than in the absence (Fig. 4A) of
extracellular Cl.
A shared property of the SLC26 anion exchangers is
cis-inhibition of the uptake of a given ion by other
substrates (19, 36, 43, 45). To assess the repertoire of
substrates capable of cis-inhibiting Cl uptake
by Slc26a6, we incubated Slc26a6-injected oocytes with sulfate,
formate, halides, nitrate, and lactate; all but lactate significantly
inhibit 36Cl uptake (Fig.
5A). A similar profile was
obtained for SO transport (Fig. 5B). We
also assessed the ability of Slc26a6 to mediate SO
exchange by measuring efflux of this tracer in the presence of
extracellular substrates (45). For this experiment,
oocytes were incubated in 35SO uptake
media for 1 h and then incubated in the absence (control group) or
the presence of the substrates indicated in Fig. 5C to see
whether these anions would transstimulate efflux of
35SO over a 30-min period. Because SLC26A4 is known to transport formate and Cl, but neither
oxalate (45) nor SO (46), we reasoned that Slc26a6 might not catalyze
formate/SO exchange, such that extracellular
formate would not stimulate efflux of
35SO. However, Fig. 5C
indicates that Slc26a6 mediates the exchange of SO with SO, Cl, formate, and oxalate;
the residual 35SO content of the
relevant oocyte groups is substantially lower than that of the control
group, indicating loss of 35SO due to
exchange with the extracellular anions. Some efflux of
35SO likely occurred in the control oocytes during the 30-min post-uptake period, because the picomoles remaining in this group (331 ± 18 pmol/oocyte) are approximately half that of the oocytes after the 1-h uptakes shown in Fig.
4A.
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Fig. 5.
Anion exchange mediated by Slc26a6. A:
cis-inhibition of 36Cl uptake in
Slc26a6-injected oocytes. Oocytes were incubated in
36Cl uptake medium for 1 h, in the
absence (control) or presence of 25 mM concentrations of the indicated
anions. B: cis-inhibition of
35SO uptake in Slc26a6-injected
oocytes. Oocytes were incubated in 35SO
uptake medium for 1 h in the absence (control) or presence of 25 mM concentrations of the indicated anions. C:
trans-stimulation of SO exchange
mediated by Slc26a6. Oocytes were incubated in
35SO uptake medium for 1 h, washed
3 times with cold uptake solution, and then incubated for 30 min in
uptake solution without 35SO in the
absence (control) or presence of 10 mM concentrations of the indicated
anions to stimulate 35SO efflux.
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As one would predict from the cis-inhibition
experiments shown in Fig. 5, Slc26a6-injected oocytes transport both
oxalate (487 ± 50 vs. 12 ± 1 pmol · oocyte1 · h1 in
water-injected controls, Fig.
6A) and formate (45 ± 5 vs. 7 pmol · oocyte1 · h1
in water-injected controls, Fig. 6B). In contrast, Slc26a1
mediates the transport of oxalate (72 ± 2 pmol · oocyte1 · h1, Fig.
6A), as reported for rat Slc26a1 (17), but not
formate (7 pmol · oocyte1 · h1, Fig.
6B).
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Fig. 6.
Oxalate and formate transport by Slc26a1 and Slc26a6.
A: oxalate uptake in oocytes injected with water, Slc26a1,
or Slc26a6. B: formate uptake in oocytes injected with
water, Slc26aA1, or Slc26a6. *Significantly different
(P < 0.01) from water-injected control oocytes.
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The activation of (rat) Slc26a1 by extracellular Cl (Fig.
4C) has been observed before (43) but not
explored in detail. To extend this observation, we first examined the
effect of several monovalent ions (halides, formate, and lactate) on
35SO uptake by Slc26a1-injected oocytes (Fig. 7A). Whereas the
divalent substrates SO and oxalate are strongly
cis-inhibitory for Slc26a1
35SO uptake (Fig. 5, A and
B), monovalent ions share activation of
35SO uptake with Cl (Fig.
7A). Although the ability of Slc26a1 to transport all of these monovalent ions has not been examined, it transports neither Cl (Fig. 4B) nor formate (Fig. 6B).
Moreover, I and Br are both
cis-inhibitory for both SO and
Cl uptake via Slc26a6, indicating that they are potential
substrates, as shown directly for SLC26A4 in the case of
I (46); by extension, these are not likely
substrates for Slc26a1. The activation of Slc26a1 by impermeant anions
is not unique to SO transport, because oxalate
transport in Slc26a1-injected oocytes is also higher in the presence of Cl and other anions (Fig. 7B). Slc26a6 serves
as a control for the latter experiment, in that oxalate transport by
oocytes injected with this cRNA is strongly cis-inhibited by
these anions (Fig. 7B).
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Fig. 7.
Differential effect of extracellular anions on Slc26a1
and Slc26a6. A: effect of extracellular anions on
SO uptake in Slc26a1-injected oocytes. Oocytes were
incubated in 35SO uptake medium for
1 h in the absence (control) or presence of 25 mM concentrations
of the indicated anions. Monovalent anions activate
35SO transport by Slc26a1, whereas they
cis-inhibit 35SO transport
by Slc26a6 (Fig. 6B). B: effect of extracellular
anions on oxalate uptake in Slc26a1-injected (open bars) and
Slc26a6-injected (filled bars) oocytes. Oocytes were incubated in
[14C]oxalate uptake medium for 1 h in the absence
(control) or presence of 25 mM concentrations of the indicated anions.
Monovalent anions activate oxalate transport by Slc26a1 and inhibit
oxalate transport by Slc26a6.
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Oocyte intracellular pH and electrophysiology.
To determine whether Slc26a6 functions as a
Cl/HCO exchanger, measurements were
made of intracellular pH (pHi) in response to the
manipulation of bath HCO and Cl. The
initial addition of CO2/HCO to
the bath solution results in the acidification of oocytes due to
CO2 plasma membrane diffusion, then intracellular hydration and dissociation, forming intracellular H+ and
HCO. Figure 8
illustrates an experiment with single water- and Slc26a6-injected
oocytes; however, these results were observed for Slc26a6-injected
oocytes from five separate frogs; rates are reported for both the
single oocytes shown in Fig. 8 and for the mean of several oocytes (in
parentheses). Figure 8 shows that a water-injected oocyte
exposed to 5% CO2/33 mM HCO (pH
7.5) acidifies by 0.44 pH units (0.46 ± 0.01, n = 8) at an initial rate of 46.0 × 104 pH units/s
(382 ± 19 × 105 pH units/s,
n = 8). The initial pHi of Slc26a6 oocytes
is essentially the same as that of water-injected control
(water-injected, 7.26 ± 0.03, n = 8; Slc26a6,
7.29 ± 0.03, n = 10); addition of 5%
CO2/33 mM HCO causes a fall in
pHi of 0.50 pH units (0.46 ± 0.02, n = 10) at an initial rate of 35.0 × 104 pH units/s (387 ± 15 × 105 pH units/s, n = 10). Slc26a6 oocytes
are depolarized (26.3 ± 4.5 mV, n = 10)
compared with control oocytes (44.8 ± 4.3 mV, n = 8). The addition of HCO causes a slight but abrupt
depolarization in Slc26a6-injected oocytes (3.1 ± 0.6 mV,
n = 9), reminiscent of the anion conductance observed in oocytes expressing NDAE1 (41). Cl
replacement (gluconate) does not affect pHi of the
water-injected control (+6.0 ± 2.2 × 105 pH
units/s, n = 8; Fig. 8A). However, Fig.
8B illustrates that Cl removal increases
pHi of a Slc26a6 oocyte at the rate of 44 × 105 pH units/s (+72 ± 8.8 × 105
pH units/s, n = 10; Fig. 8B), which ceases
after Cl readdition. Surprisingly, this gluconate
replacement evokes a 37-mV hyperpolarization (22.7 ± 2.9 mV,
n = 9; vs. +0.2 ± 2.0 mV, n = 8 for controls). A second Cl removal in Fig. 8B
alkalinizes the cell at 28 × 105 pH units/s
(+41 ± 6.2 × 105 pH units/s,
n = 8) and reproduces the hyperpolarization
(18.6 ± 3.8 mV, n = 8). In all the experiments
with Slc26a6-injected oocytes, the second alkalinization induced by
Cl removal, which occurs at a higher pHi, has
a lower rate (+72 × 105 pH units/s for the first
alkalinization and +41 × 105 vs. +6.0 × 105 pH units/s for the single Cl removal in
water-injected oocytes).
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Fig. 8.
HCO transport mediated by Slc26a6, characterized
using ion-selective microelectrodes. A: experiment
monitoring intracellular pH (pHi) and membrane potential
(Vm) of a water-injected oocyte. Addition of 5%
CO2/33 mM HCO (pH 7.5) to the bath
elicited a slight depolarization and results in cell acidification;
CO2 diffuses across the membrane, forms
H2CO3, and then subsequently generates
H+ and HCO from fast dissociation.
B: experiment monitoring pHi and
Vm of a Slc26a6 oocyte. The initial pH and rate
of CO2-induced acidification are equivalent to those of the
water-injected control. In the continuing presence of 5%
CO2/33 mM HCO (pH 7.5),
Cl removal elicits a robust alkalinization that halts
with Cl readdition. Simultaneously, there is a pronounced
and reversible hyperpolarization not observed in control oocytes.
Replacement of Na+ (choline) elicits no change in
( )pHi and a small hyperpolarization as observed in
control cells. A second Cl removal elicits a lesser
alkalinization and a second hyperpolarization.
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We also replaced Na+ with choline to test cation dependence
of Slc26a6. Na+ removal and replacement did not obviously
affect pHi. Before CO2 removal, pHi
rose to 7.2, i.e., almost the non-HCO level.
Finally, removal of 5% CO2/33 mM HCO elicits a robust alkalinization and pHi overshoot to 7.9 (7.83 ± 0.07, n = 10), indicative of cellular
HCO loading [change in ()pHi for
Slc26a6 is +0.53 ± 0.07, n = 10] (40,
41). This overshoot is not observed in controls
(pHi for controls is +0.02 ± 0.04, n = 8) (40, 41).
Because Slc26a6 clearly functions as a
Cl/HCO exchanger (Fig. 8B),
we tested whether it could function as a
Cl/OH exchanger. For these experiments, we
continuously bubbled all the non-HCO solutions with
100% O2 as previously described in the characterization of
NDAE1 (41). Figure 9
illustrates the non-HCO responses of one control and
one Slc26a6 oocyte; the data for these two oocytes are detailed in the
legend for Fig. 9. Data for the mean of several oocytes and several
frogs are written in parentheses. Bath Cl removal of
control oocytes (Fig. 9A) does not change pHi
(2.1 ± 1.8 × 105 pH units/s,
n = 7) or membrane potential (+1.1 ± 1.7 mV,
n = 14). Nevertheless, this same maneuver alkalinized
(+27 ± 6.4 × 105 pH units/s,
n = 6) and hyperpolarized (9.1 ± 3.0 mV,
n = 11; P < 0.008) Slc26a6 oocytes
(Fig. 9B). Cl readdition to the bath stopped
the alkalinization and returned membrane potential to the initial
value. However, the readdition of Cl evoked a large
transient depolarization (+39.6 ± 4.9 mV, n = 11 for Slc26a6 vs. 4.6 ± 0.9 mV, n = 14 for
controls; P < 0.000002) followed by a smaller
sustained depolarization.
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Fig. 9.
Slc26a6 mediates Cl/OH
exchange in Xenopus laevis oocytes. A:
experiment monitoring pHi and Vm of
a water-injected oocyte. Removal of bath Cl does not
alter pHi (rate pHi = 0.0 × 105 pH units/s) and minimally changes
Vm (0 Cl
Vm = 9.9 mV). Similarly, readdition of
Cl elicits minimal pHi
(pHi = 0.00) and Vm changes
(plus Cl Vm = +2.7 mV).
B: experiment monitoring pHi and
Vm of an Slc26a6-injected oocyte is shown. For
this experiment, the initial baseline pH (7.23) is slightly elevated
compared with the water-injected control (pHi = 7.19, Fig 9A). In the nominal absence of HCO
(O2 bubbling), Cl removal elicits an
alkalinization (pHi = 0.07 pH units; rate
pHi = +23.0 × 105 pH units/s)
that halts with Cl readdition. Simultaneously, there is a
reversible hyperpolarization (0 Cl
Vm = 13.0 mV) with a pronounced
depolarization induced by the readdition of Cl (plus
Cl Vm = +45.7 mV).
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DISCUSSION |
We report here the molecular and functional characterization of
Slc26a1 and Slc26a6, the first and sixth members of the murine Slc26
gene family of anion exchangers. We have also reported on the
chromosomal localization and genomic structure of the two genes, along
with evidence of alternative splicing of both transcripts. Immunolocalization and functional characterization recently identified Slc26a6 as the putative Cl/formate exchanger of the renal
proximal tubular epithelium (19), and our data indicate
that Slc26a6 can also mediate SO exchange,
Cl/oxalate, Cl/OH, and
Cl/HCO exchange. Basolateral
oxalate-SO/HCO exchange in the
proximal tubule is thought to be mediated by Slc26a1/SLC26A1 (Sat-1)
(17), and a direct functional comparison indicates that SO and oxalate exchange by these homologous
transporters is mechanistically distinct.
The sequence comparison of mouse Slc26a6 reveals that the closest
characterized relative is prestin (Slc26a5), with 39% identity at the
amino acid level, whereas Slc26a1 is closest to Slc26a2 (DTDST, 47%
identity). The COOH-terminal domains of both Slc26a1 and Slc26a6
contain predicted sulfate transporter and anti-sigma (STAS) domains,
recently defined by virtue of homology between the SLC26
gene family and bacterial anti-sigma factor antagonists (2). Structural predictions suggest a role for the STAS
domain in nucleotide binding and/or hydrolysis (2). With
the exception of a COOH-terminal type I PDZ interaction motif
(51) (see below), analysis of the Slc26a6 protein with
motif-based algorithms (44, 60) does not reveal other
protein-signaling domains or motifs. Mouse and rat Slc26a1
(4) predict a COOH-terminal type I PDZ binding motif
(S-A-L); however, this motif is not conserved in the human protein
(GenBank AF297659). Analysis using both the Prosite and Phosphobase
(22) databases reveals that Slc26a1 and Slc26a6 are both
potential substrates for a number of protein kinases, including
tyrosine kinases, protein kinase A, and protein kinase C (Fig. 1).
We utilized heterologous expression in X. laevis oocytes for
the functional characterization of Slc26a1 and Slc26a6. Isotopic flux
studies indicate that Slc26a6 is a versatile anion exchanger, capable
of transporting SO, Cl, formate, and
oxalate, whereas Slc26a1 transports only SO and
oxalate in flux assays. The direct measurement of changes in
pHi elicited by the removal of extracellular
Cl in both the presence and absence of
HCO indicates that Slc26a6 functions as a
Cl/HCO and
Cl/OH exchanger (Figs. 8 and 9). While this
manuscript was in review, Wang et al. (58) reported that
Slc26a6-injected X. laevis oocytes can mediate
Cl/HCO exchange. The lack of
stimulation of 36Cl uptake by Slc26a6 by a
more acidic extracellular medium (Fig. 4B) is somewhat
surprising, given that this protein can mediate Cl/OH exchange (Fig. 9). However, similar
findings for the pH dependency of Slc26a6 Cl uptake were
reported by Knauf et al. (19).
Slc26a6 shares the ability to mediate Cl/base exchange
with Slc26a3 (34) and Slc26a4 (42, 49).
However, the electrophysiology of this mode of anion transport has not
been studied in the SLC26 anion exchangers. We have found that removal
of extracellular Cl in the presence of
HCO evokes a significant hyperpolarization. The
simplest explanation is that Slc26a6 mediates electrogenic
HCO transport. That is, these data are consistent
with 1) cation (e.g., H+) coexit with
Cl; 2) the entry of another anion with
HCO into the cell; or 3) exchange of more
than one HCO ion for each Cl ion that
exits. We cannot rule out the possibility that Slc26a6 overexpression
somehow evokes an otherwise silent conductance natively present in the
oocyte, and future experiments will examine the nature of these
Cl-suppressed voltage or current changes. However,
unmasking an otherwise silent conductance in response to Slc26a6
expression seems unlikely, because there is not a precedent in the
literature for a "Cl-inhibited anion current" as
measured in our experiments.
Although complete functional characterization is lacking for many of
the SLC26 anion exchangers, the range of substrates intrinsic to
Slc26a6 is tentatively only matched by that of SLC26A3 (DRA) (5,
34, 36, 54). Of note, however, formate transport has not
been reported for SLC26A3, and only low-level oxalate, Cl, and SO uptake compared with
water-injected controls has been reported for SLC26A3-injected oocytes
(36, 48). In contrast to Slc26a6, Slc26a1 does not
transport Cl or formate but does transport
SO and oxalate (Figs. 4-7). SLC26A2, in turn,
is known to transport monovalent anions such as Cl,
I, and formate, but not divalent anions such as
SO and oxalate (45, 46). This
combination of functional divergence and structural homology within the
SLC26 gene family will no doubt aid the structure-function analysis of
anion specificity.
The data reported here reveal that transport of the divalent anions
SO and oxalate by Slc26a1 is strongly activated by
impermeant, monovalent anions such as Cl and formate.
This is, presumably, not simply a matter of the valence of anionic
charge, because HCO reportedly does not activate
SO uptake by rat Slc26a1 (43). The
physiological significance of this phenomenon is not yet clear;
however, this functional characteristic of Slc26a1 is dramatically
different from that of other SLC26 anion exchangers, including Slc26a2
(43). It is hoped that a comparative structure-function
analysis of Slc26a1 and Slc26a2 will yield some insight into the
molecular mechanism. As shown in Fig. 4, SO
exchange by Slc26a1 is significantly stimulated by an acid-outside pH
gradient, particularly in the presence of extracellular
Cl (Fig. 4C). This is suggestive of
H+-SO cotransport, as reported for AE1
(reviewed in Ref. 6), a member of the
HCO transporter superfamily (SLC4) that can also
mediate the transport of SO, Cl, and
HCO.
The finding that Slc26a6 functions in the exchange of formate and
oxalate for both Cl and SO is of
particular relevance to its role in the kidney. In conjunction with
Na+/H+ exchange mediated by NHE3, apical
Cl/formate exchange functions in transepithelial
reabsorption of NaCl by the proximal tubule (57) and some
segments of the distal nephron (55). The
perfusion of proximal tubules with luminal oxalate also stimulates
transepithelial salt transport, although in this case
Na+-SO cotransport rather
than Na+/H+ exchange absorbs Na+
(see below) (56). An important observation is that Slc26a6 mediates both Cl/formate and Cl/oxalate
exchange, given that previous studies using renal brush-border vesicles
suggested the existence of two separate distinct transporters, one
capable of only Cl/formate exchange and the other capable
of both Cl/formate and Cl/oxalate exchange
(16). The Cl/formate/oxalate exchanger
activity in brush-border vesicles has a higher affinity for oxalate
over formate (16); although kinetic studies of Slc26a6 are
lacking, the greater efficacy of oxalate in both
cis-inhibition of 36Cl uptake
(Fig. 5A) and trans-stimulation of
35SO efflux (Fig. 5C) are
consistent with this property. Unlike Cl/formate
exchange, Cl/oxalate exchange in apical membrane vesicles
is thought to be electrogenic (16), compatible perhaps
with the observation that Cl/HCO
exchange by Slc26a6 is not electroneutral (Fig. 8). The question of
whether two separate gene products mediate Cl/formate
exchange in the proximal tubule awaits the characterization of Slc26a6
knockout mice, along with the full pharmacological characterization of
the Slc26a6 in its various transport modes (16). A second
unresolved issue is how the luminal exchange of Cl with
formate and oxalate is specifically coupled to
Na+/H+ exchange and
Na+-SO cotransport,
respectively (56). This lack of physiological coupling
between luminal formate and apical
Na+-SO cotransport remains unexplained, because Slc26a6 can evidently mediate both
SO/formate and Cl/formate exchange
(Fig. 5).
Finally, another longstanding issue in the transcellular transport of
NaCl by the proximal tubule has been the relative importance of apical
Cl/f
TOP
ABSTRACT
INTRODUCTION
