1 Departments of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555; and 2 Laboratoire Jean Maetz, Centre National de la Recherche Scientifique and Commissariat à l'Energie Atomique Unité de Recherche Associée 1855, 06320 Villefranche-sur-Mer, France
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ABSTRACT |
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Cl channels are
important for ion transport and cell volume regulation in A6 renal
cells. In the present study, we used reverse transcriptase
(RT)-polymerase chain reaction (PCR) and rapid amplification of cDNA
ends (RACE) to identify proteins homologous to ClC
Cl
channel proteins in A6
cells. Using degenerate primers designed on consensus sequences for
members of the ClC family, we amplified an RT-PCR product that had
significant homology to the ClC sequences. RACE-PCR was then used to
isolate several full-length clones that had total lengths from 2,764 to
3,016 base pairs. Although the coding regions were identical, sequence
differences occurred in the 5' noncoding regions. The amino acid
sequences of the clones had high homologies to rat and human ClC-5 (85 and 84%, respectively, if the 5th methionine of the open reading frame
represents the start codon). Three parts of the protein (53, 80, and 63 amino acids in length) were 97-100% homologous to the mammalian
sequences. Ribonuclease protection assay analysis revealed mRNA for
this protein in oocytes, kidney, intestine, liver, brain, and blood, with lower amounts in stomach, muscle, and skin. Expression of the
clones in Xenopus
laevis oocytes resulted in an
outwardly rectifying Cl
current that was inhibited by
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and
possessed an anion selectivity of
I
> Cl
>> gluconate.
cultured renal cells; outwardly rectifying chloride current; ClC-5; Xenopus laevis
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INTRODUCTION |
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THE CULTURED RENAL epithelial cell line A6 forms
well-differentiated monolayers that have properties similar to renal
distal tubule epithelia. Although A6 cells have been most frequently used as a model of transepithelial
Na+ transport, these cells are
also advantageous for investigations of
Cl channels. Several types
of Cl
channels that have
been implicated in either transepithelial Cl
transport or cellular
homeostasis have been described in the apical and basolateral membranes
of polarized A6 monolayers. For example, Marunaka and Eaton (19)
reported 3-pS and 8-pS Cl
channels in the apical membrane that may provide a route for Cl
secretion following
hormonal stimulation mediated by adenosine 3',5'-cyclic
monophosphate (cAMP) (14, 32).
Cl
channels are also
involved in the transepithelial
Cl
reabsorption that occurs
in parallel with Na+ transport and
is stimulated by cell swelling (6a). In the basolateral membrane,
patch-clamp studies by Banderali and Ehrenfeld (2) have identified four
different types of Cl
channels. Three present linear current-voltage
(I-V)
relationships with unitary conductances of 12 pS, 30 pS, and 42 pS,
whereas the fourth shows an outwardly rectifying
I-V
relationship with inward and outward conductances of 16 pS and 57 pS,
respectively. All of these channels were activated by hyposmotic
bathing solutions.
Little is known about the molecular identity of the A6
Cl channels, although
recently the cystic fibrosis transmembrane conductance regulator has
been cloned from A6 cells by Price et al. (21). This channel has
properties similar to the apical 8-pS channel described by Marunaka and
Eaton (19). The purpose of the present work was to determine whether
members of another Cl
channel family, ClC channels, were present in this cell line. This
rapidly growing group of voltage-gated
Cl
channels includes
several members that are expressed preferentially in renal epithelial
cells (9). At least one ClC member, ClC-2, is activated by hypotonicity
(11, 27).
In the present study, we used a polymerase chain reaction (PCR) cloning
strategy to isolate proteins homologous to members of the ClC family in
A6 Xenopus
laevis renal cells. We now report a
novel ClC sequence, Xenopus ClC-5
(xClC-5), that has strong homology to mammalian ClC-5. The xClC-5 gene
is most abundantly expressed in kidney, intestine, and oocytes.
Nonetheless, injection of Xenopus
oocytes with xClC-5 cRNA leads to the expression of a novel
Cl conductance, not
observed in control (water-injected) oocytes, that is blocked by
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and
has a selectivity sequence of
I
> Cl
>> gluconate.
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MATERIALS AND METHODS |
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A6 cell culture. A6 cells from X. laevis kidney (American Type Culture Collection) were grown according to the method of Wills et al. (30, 31). Briefly, cells from passages 74-79 were grown in specific culture medium (GIBCO 84-5022 supplemented with antibiotics and 10% fetal calf serum; HyClone Laboratories, Logan, UT) on plastic flasks or on permeable filter supports (Transwell 3419; Costar) at 28°C in an atmosphere of 1% CO2 in air.
Isolation of mRNA. Total RNA was prepared from A6 cells or X. laevis tissues following the method of Chomczynski and Sacchi (6). Organs were removed from anesthetized animals and either extensively washed or perfused with saline buffer to remove most of remaining blood (heart, liver, muscle, brain, kidney, oocytes) and contents (stomach, urinary bladder) or further dissected to prepare different epithelial layers (intestine, skin). For A6 cells, poly(A)+ RNA was selected on oligo(dT) cellulose columns from the total RNA preparation (1) and analyzed by agarose gel electrophoresis. The average final yield was 15 µg/106 cells for the total RNA and 130-180 ng/106 cells for poly(A)+ RNA.
Reverse transcriptase-PCR. Degenerate
sense (U) and antisense (D) oligonucleotide primers were designed from
consensus sequences of several
Cl channel sequences of
members of the ClC family (ClC-0, -1, -2, and -K) as follows: U,
GT(C/G) CTN TT(C/T) AG(C/T) AT(A/T/C) GA(G/A) GTN A; and D, AT(C/T) TGN
CCN GTN A(G/A)(C/T) TC(G/A) AA. For first-strand synthesis, 0.5 µg of
A6 poly(A)+ RNA was reverse
transcribed using Moloney murine leukemia virus reverse transcriptase
(RT) H
(Stratagene, La
Jolla, CA) at 37°C for 20 min. After incubation at 70°C for 15 min, ribonuclease (RNase) H was added and the reactions were kept again
at 37°C for 20 min. The RT reaction (1-3 µl) was used
directly for amplification. The PCR solution was assembled and heated
at 94°C for 3 min before Taq DNA
polymerase (Stratagene) was added. Subsequently, PCR was performed
using the following profile: 55°C for 1 min, 72°C for 2 min,
and 94°C for 45 s, for 40 cycles. The last cycle was terminated
after an elongation time of 8 min. The RT-PCR products were gel
purified, cloned into the PCR II vector (Invitrogen), and partially
sequenced.
Rapid amplification of cDNA ends. The
PCR template used for rapid amplification of cDNA ends (RACE) was
generated through a cDNA amplification kit (Marathon, Clontech, Palo
Alto, CA) according to the supplier's recommendations. Briefly,
poly(A)+ RNA was converted into
double strand cDNA, ligated to a cDNA adaptor, diluted 100-fold, and
denaturated by heating at 95°C for 5 min. Aliquots were stored at
20°C. Five microliters at a time were used for a PCR
reaction of 50 µl final volume. Amplification was carried out using a
polymerase mix containing thermostable Taq and
Pwo DNA polymerases (Expand Long
Template PCR System, Boehringer Mannheim, Mannheim, Germany). Both the
5' and the 3' RACE reactions were primed with an internal
gene-specific primer and the Marathon adaptor primer. The PCR reactions
were performed under the following conditions: denaturation at 94°C
for 45 s, annealing at 60°C for 30 s, and elongation at 68°C
for typically 1-2 min, depending on the expected size of the
fragment [1 min/kilobase (kb)], for 35 cycles. Before
addition of the polymerase mix, the solution was heated for 3 min at
94°C. The last cycle was terminated after an elongation step of 8 min at 68°C. The RACE products were gel purified, cloned into the
expression vector pGEM-5Zf(+) (Promega, Madison, WI), and sequenced.
To acquire the maximum sequence information for the 5' end, two independent PCR were performed and the 5' ends of seven separate clones of the 5' RACE products were sequenced. Full-length cDNAs were obtained by standard long-distance PCR with gene-specific primers from the 5' and the 3' ends, using the adaptor-ligated A6 cDNA.
cDNA sequencing. In general, cDNA was sequenced by the chain termination method of Sanger et al. (25). Selected RACE products as well as full-length clones were sequenced in both directions, using an Applied Biosystems model 373A sequencing unit with synthetic primers (Recombinant DNA Laboratory, University of Texas Medical Branch). The BLASTN database search program (National Center for Biotechnology Information) was used to identify homologies of the obtained sequences to published sequences. Putative transmembrane domains in the sequence data were predicted using the algorithm of Kyte and Doolittle (16).
Northern blot analysis. A6 poly(A)+ RNA (10 µg) was resolved on a 1.2% agarose gel under denaturing conditions and transferred overnight in 10× saline sodium citrate (SSC) onto a nylon membrane (Hybond N+, Amersham, Buckinghamshire, UK). After the RNA was ultraviolet-cross-linked to the membrane, the blots were hybridized for 3 h at 65°C in rapid-hybridization buffer (Amersham) to a xClC-5 cDNA probe labeled with [32P]dCTP (Rediprime labeling kit, Amersham). The membranes were washed with a final stringency of 0.1% SSC-0.1% sodium dodecyl sulfate at 65°C. Hybridization was visualized by autoradiography.
Antisense RNA probe synthesis. The
plasmid containing the full-length xClC-5 cDNA was digested with
Sst I, gel purified, and religated.
The resulting construct was linearized by digestion with
Pvu II. From the linearized plasmid, a
radiolabeled antisense RNA fragment with a length of 257 bases, 34 bases of the polylinker region and 223 bases complementary to the
xClC-5 sequence, was produced. For in vitro transcription, SP6
polymerase (10 units) and 50 µCi
[-32P]UTP (10 mCi/ml, 800 Ci/mmol) were used (MAXIscript, Ambion, Austin, TX),
following the protocol given by the manufacturer. The DNA template was
removed by digestion with RNase-free deoxyribonuclease (4 units) at
37°C for at least 20 min. After addition of an equal volume of
gel-loading buffer, the solution was heated at 95°C for 3 min and
purified by electrophoresis on a 5% polyacrylamide gel containing 8 M
urea. The radiolabeled RNA runoff was localized by autoradiography,
excised, and eluted overnight at room temperature in 350 µl of
elution buffer. This solution has been stored at
20°C
without removal of the gel fragment.
To generate an RNA probe that can be used as an internal control, a 751-base pair (bp) fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified by PCR from A6 cDNA and cloned. After digestion with Apa I, the above-described procedure was carried out. The labeled full-length antisense RNA probe was 244 bases long, with 169 bases complementary to the cloned GAPDH.
RNase protection assay. For the RNase protection assay (RPA), an RPA II kit (Ambion) was used. The experimental procedure followed the recommendations of the supplier. The xClC-5 and GAPDH antisense RNA probes (0.3-1.0 × 105 counts/min) were incubated with 10 µg of total RNA from various X. laevis tissues. The hybridization mixture was heated at 95°C for 3 min and incubated at 44°C overnight. After hybridization, single-stranded RNA was digested with 0.13 units of RNase A and 5.3 units of RNase T1 at 37°C for 40 min. Protected RNA was precipitated, resuspended, and electrophoresed through a 5% polyacrylamide-8 M urea gel. The migration was visualized by autoradiography. The sizes of the protected fragments were determined with respect to 32P-labeled RNA molecular mass markers (Sigma, St. Louis, MO).
Functional expression in Xenopus
oocytes. With use of T7 RNA polymerase, capped cRNA was
prepared from the pGEM-5Zf(+) cloning vector containing the xClC-5
sequence and stored at 80°C in diethyl pyrocarbonate-treated
water at a final estimated concentration of 50 ng/µl. cRNA
(2.5-5 ng) was injected into X. laevis oocytes prepared and handled as previously
described (22). After 3-4 days, at 18°C, the oocytes were
investigated by two-microelectrode voltage clamping using a TEV 200 amplifier (Dagan, Minneapolis, MN) monitored by computer through a
Digidata 1200 analog-to-digital converter and pCLAMP software (Axon
Instruments, Foster City, CA). Microelectrodes were pulled using a
Zeitz puller (Augsburg, Germany), were filled with a 3 M KCl solution,
and had resistances of 1.5-2.5 M
. Oocytes were voltage clamped
at a holding potential of
50 mV, and 800-ms voltage steps from
100 to +80 mV in 20-mV increments were applied. All experiments
were performed at room temperature. Oocytes were perfused with an
experimental medium that contained (in mM) 95 NaCl, 2 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2,
0.41 CaCl2, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 3 NaOH (pH 7.4). In ion substitution experiments, 80 mM
Cl
was replaced with an
equal concentration of gluconate or
I
, and 3 M KCl agar-agar
bridges were used to minimize junction potentials (6-8 mV). The
relative permeability of I
or gluconate vs. Cl
was
estimated using the following expression derived from the Goldman-Hodgkin-Katz equation
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(1) |
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RESULTS |
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Cloning of an amphibian renal ClC
homologue. RT-PCR was performed using A6 cell mRNA and
degenerate oligonucleotide primers designed from consensus sequences of
several Cl channel
sequences of members of the ClC channel family (ClC-0, -1, -2, and -K;
see MATERIALS AND METHODS). A PCR
product with an expected length of 830 bp was obtained. Sequence
analysis revealed that the amplified cDNA had significant homology to
the sequences of ClC-2 and ClC-K. The 5' and 3'
extremities of the new cDNA were successfully isolated by RACE-PCR,
which allowed the subsequent amplification of full-length clones in
long-distance PCR using clone-specific primers. As shown in Fig.
1, the longest of
the obtained cDNAs consisted of 3,016 bp with an open reading frame of
2,424 bp that was preceded by a 435-bp 5' noncoding region and
followed by a 157-bp 3' noncoding region. The initiation Met was
assigned to the first ATG codon in-frame that was preceded by an
in-frame stop codon. cDNAs of three clones (5A2, 5A6, 5B1) from two
separately performed amplification reactions were sequenced completely
in one direction, and one of the clones (5A2) was sequenced in both
directions. Comparison of the three coding sequences showed that clone
5A2 has an adenosine residue at position 585 instead of a guanosine
residue (as in clones 5A6 and 5B1), which does not result in an amino
acid difference. Only clone 5A6 had a nucleotide difference that
resulted in an amino acid substitution. Specifically, there was a T/A
exchange at position 1276, such that amino acid 426 was Ser instead of
Thr in the other two sequences. Whether these differences were due to
the existence of two different DNA sequences or to misincorporations of
bases during cDNA synthesis or during the amplification reaction has
not been studied. However, these results clearly show the high fidelity
of the Pwo DNA polymerase used in the
PCR reactions and the advantages of the RACE cloning strategy.
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Interestingly, we found that, although the clones possessed identical
coding and 3' noncoding sequences, there were several differences
in their 5' noncoding regions. Compared with the sequence of
clone 5A2 (Fig. 2), the 5'
untranslated sequences of clone 5A6 and 5B1 are not only shorter at
their 5' extremities but show specific deletions within their
sequences [from nucleotides 121 to
268 (5A6) and
217 to
269 (5B1)]. Because the length and composition of the 5' noncoding region are known to be important for the translation efficiency of mRNA in vivo (10), we injected cRNAs
of all three clones into Xenopus
oocytes. Three batches of oocytes were injected with cRNA for clone 5A2
(n = 10), and one batch of oocytes was
injected with cRNA for clones 5B2 and 5B1
(n = 2 and
n = 5, respectively). No qualitative
differences in expression were detected among the clones. The
experiments reported below were performed using clone 5B1.
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The open reading frame predicts an 808-amino acid translation product
with a molecular mass of 90 kDa. Figure 3
shows a hydrophobicity analysis of the predicted amino acid sequence
using the method of Kyte and Doolittle (16). Like that of other members
of the ClC Cl channel
family, the hydrophobicity profile of this channel shows 10-12
hydrophobic regions, indicated as numbered lines above the sequence
data in Fig. 4. Three potential
N-glycosylation sites have been found
at positions Asn-17, Asn-169, and Asn-470. The latter is located
between hydrophobic regions D8 and D9 and represents a highly conserved
glycosylation motif among all ClC channels except for ClC-7. In vitro
translation experiments by Kieferle et al. (15) showed that the
corresponding segments of ClC-0, -1, -2, and -K1 are
glycosylated. Consequently, it has been suggested that this segment is
positioned at the extracellular side of the cell membrane. Likewise,
Asn-169 is located in a predicted extracellular loop between
hydrophobic regions D1 and D2. Because Asn-17 is located close to the
amino terminus, and therefore the cytoplasmic part of the protein, it
is not very likely that this amino acid is glycosylated in vivo
[see Ref. 17 for a putative transmembrane model of human (h)
ClC-5]. Consensus sequences for cAMP- and guanosine 3',5'-cyclic monophosphate-dependent protein kinase
phosphorylation are present at positions Ser-10, Thr-411, and Thr-412.
Protein kinase C consensus sites were found at positions Thr-353,
Thr-411, Ser-459, Thr-606, Ser-639, Ser-690, Ser-708, Thr-719, Ser-738, and Thr-786. As in other ClC proteins (except ClC-7), the Glu at
position 234 and Pro at position 513 were conserved.
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The ClC family has rapidly grown to eight members, at present, which
can be classified into three subfamilies as follows: 1) ClC-0, -1, -2, -K1, and -K2;
2) ClC-3, -4, and -5; and
3) ClC-6 and -7. Analysis of the
full-length sequence of xClC-5 revealed its high homology to the more
recently isolated group of ClC
Cl channels, the outwardly
rectifying Cl
channel
(ORCC) subbranch comprising ClC-3, -4, and -5. The protein sequence of
xClC-5 has a 69% homology to hClC-3 (3), 73% to hClC-4 (29), and 77%
to hClC-5 (Ref. 7; see Fig. 4). The homologies to other mammalian ClC
proteins are 72% to rat (r) ClC-3 (13), 74% to rClC-4 (T. J. Jentsch,
W. Günther, M. Pusch, and B. Schwappach, GenBank no. Z36944;
direct submission), and 77% to rClC-5 (24, 26). In contrast, the
homology was only 19.8% to rClC-2 (27) and 14.5% to hClC-6 (4). If
Met-65 (i.e., the 5th Met of the open reading frame) is assigned as the initiation ATG, the homology to hClC-5 increases to 84% (and for rClC-5 to 85%). Because of the high homology to the hClC-5 and rClC-5
sequences, the identified clone has been named xClC-5.
In Northern blot analysis (Fig. 5), a full-length xClC-5 cDNA probe hybridized to a band at ~3.4 kb. As shown in Fig. 6, RPA of a variety of Xenopus tissues showed that xClC-5 is a broadly expressed gene. mRNA was mostly present in oocytes, kidney, and intestine. Lower amounts were found in liver, blood, brain, heart, and urinary bladder. Very low but detectable levels were discovered in stomach, muscle, and skin. An antisense probe for GAPDH mRNA detection was included in each sample, serving as internal control for RNA integrity. The variation in GAPDH mRNA levels in the different tissues was consistent with the tissue-specific GAPDH gene expression described by Piechaczyk et al. (20). Two fragments of protected GAPDH mRNA were present in every analysis. Because these two fragments have been found only in samples to which antisense GAPDH mRNA probe has been added (data not shown), we can exclude the possibility that one of the fragments was mRNA protected by xClC-5 antisense probe. The existence of more than one protected GAPDH fragment was expected, since sequencing of several partial A6 GAPDH cDNA sequences (obtained by PCR) revealed that at least two isoforms were present in A6 cells. One isoform was the same as that found for GAPDH protein from A6 cells by O. K. Al-Khalili and D. C. Eaton (GenBank no. U41753; direct submission).
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Electrophysiological characteristics of expressed
xClC-5. The membrane potential
(Vm) of control
oocytes had a mean value of 66 ± 6 mV
(n = 10). As illustrated in Fig.
7C
(water-injected oocytes), the currents applied to clamp the oocyte
membrane were low over the measured potential range (<300 nA at +80
mV and less than
50 nA at
100 mV). The
Vm of xClC-5
cRNA-injected oocytes (4 days after the cRNA injection) were
significantly (P < 0.0001) depolarized compared with controls, with a mean
Vm of
23 ± 1 mV (n = 29; 4 frogs). As shown
in the example presented in Fig. 7, B
and C, cRNA-injected oocytes showed a
large outwardly directed current at positive holding potentials.
Partial substitution of Cl
in the bathing solution with 80 mM gluconate led to a decrease of the
current amplitude in the positive voltage range, as expected for a
relatively large
Cl
-to-gluconate
permeability ratio (Fig.
8A). The
resting potential shifted from
21 ± 2 mV to
7 ± 2 mV [change in
Vm
(
Vm) =
14 ± 3 mV; n = 4;
P < 0.05] following partial
substitution of Cl
with
gluconate and from
23 ± 3 mV to
30 ± 5 mV
(
Vm = 8 ± 1 mV; n = 4;
P < 0.025) after partial
substitution of Cl
with
I
. The permeability ratios
calculated using Eq. 1 were
Pgluconate/PCl = 0.5 ± 0.2 and
PI/PCl = 1.4 ± 0.1. Consequently, the anion selectivity sequence was
PI > PCl > Pgluconate.
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Application of the Cl
channel blocker DIDS (500 µM) to cRNA-injected oocytes held at +80 mV
reduced the clamping current from 11,095 ± 2,381 nA to 2,864 ± 662 nA (Fig. 8B;
n = 5). The inhibitory effect was
voltage dependent, since it was stronger at positive potentials (72.2 ± 7.3% at +80 mV) than at negative potentials (31.9 ± 8.1% at
100 mV). The inhibition was not reversible after a 6-min
perfusion period.
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DISCUSSION |
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The present study describes the first cloning and functional expression
of a ClC Cl channel
isolated from the renal epithelial cell line A6. The longest complete
cDNA had 3,016 nucleotides and contained a 5' untranslated
sequence of 435 nucleotides, a 3' untranslated sequence of 157 nucleotides, and an open reading frame of 2,424 nucleotides coding for
an 808-amino acid protein. The first initiation Met is preceded
by an in-frame stop codon. The amino acid sequence shows a
high homology to rClC-5 and hClC-5, members of the ClC family that is
associated with the genetic disorder Dent's disease (7, 8, 17). The
homology to both of these clones increases to 85 and 84%,
respectively, if the first amino acid of the sequence is assigned to
Met-65. Relatively poor homology was found to other ClC
Cl
channels except for
ClC-3 and ClC-4. Therefore, this new channel, named xClC-5, can be
considered a member of the ORCC subbranch of the ClC
Cl
channel family.
Structural properties of xClC-5. An alignment of the xClC-5 amino acid sequence with the hClC-5 sequence (Ref. 8; Fig.4) revealed several structural features that are common to all members of the ClC family. The N-glycosylation motif between D8 and D9 was highly conserved, as were the sites for putative phosphorylation (see Fig. 1). An additional putative glycosylation site was found in a predicted extracellular loop at Asn-169, and an additional putative protein kinase A phosphorylation site is present at Ser-10. As in ClC-2 (11), domains necessary for channel activation could be present in the amino terminus of the sequence, where Ser-10 could offer a target sequence for regulatory mechanisms. As in other ClC members, the hydrophobic domain D13 was also found. It is remarkable that the sequence homology for the 53 amino acids of the cytosolic carboxy terminus (which includes region D13) was 100% for the xClC-5 and the hClC-5 sequences. This finding may indicate the importance of this segment for channel function or regulation. Amino acids 293-373, a fragment of 80 amino acids that includes hydrophobic domains D5 and D6, constituted another entirely conserved part of the amphibian sequence compared with the human sequence. A high homology (97%) was also found for a segment of 63 amino acids from position 553 to position 616, comprising D11 and D12.
The size of the xClC-5 mRNA obtained by Northern blot analysis was ~3.4 kb. This value was consistent with the length of the obtained clones (2.7-3.0 kb), assuming that an additional portion is created by polyadenylation. However it was notably smaller than that found for the mammalian transcripts of ClC-5. hClC-5 and rClC-5 probes both recognized messages of 9.5 kb (8, 24, 26), but the open reading frames encoding the respective proteins were confined to a portion of only 2,238 bp. Similar findings have been obtained for hClC-4 (29), in which an open reading frame of ~2.2 kb was found at the 3' end of a 7.5-kb message. Consequently, these authors suggested the existence of large 5' noncoding regions in the mammalian sequences. Apparently, the amphibian clone is lacking these large 5' untranslated regions.
Previous studies using Northern blot analysis have shown that rClC-5 and hClC-5 were predominantly expressed in kidney (7, 24, 26), although mRNA has also been detected in rat brain, liver, lung, and intestine. In the present study, RPA showed that xClC-5 is a rather broadly expressed gene that is most highly transcribed in oocytes, kidney, and intestine. xClC-5 mRNA is also present in liver, blood, brain, heart, and urinary bladder. Significant, lower but detectable amounts were found in stomach, muscle, and skin. Considering the high sensitivity of the RPA technique, a contamination caused by mRNA of erythrocytes remaining in capillary blood vessels of the analyzed tissues cannot be excluded in muscle, heart, kidney, and liver, where complete perfusion was difficult to obtain. To our knowledge, there is no previous report of the presence of any member of the ClC family in erythrocytes. It will be interesting to determine whether xClC-5 is present only in nucleated erythrocytes like those from X. laevis or whether this protein can be found as well in mammalian erythrocytes that lack a nucleus.
Functional properties of xClC-5.
Injection of xClC-5 cRNA in Xenopus
oocytes induced the appearance of outwardly rectifying Cl currents that were not
found in control oocytes. This current had a selectivity sequence of
I
> Cl
>> gluconate and was
blocked by DIDS. Outward rectification has been described for hClC-5
and rClC-5, also expressed in Xenopus oocytes (8, 26), and has been interpreted as an open channel rectification or a voltage dependence of a gate with fast kinetics (26). The currents induced by rClC-5 (26) were time independent, similar to our results. The permeability ratio (based on changes in
reversal potential) was not given for the rat study (26); however, the
reported (conductance) selectivity sequence was
> Cl
> I
and, therefore, differs
from our study. Cl
channel
blockers such as DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid, or diphenylamine-2-carboxylic acid had no effect
(26). In contrast, Sakamoto et al. (24), who used rClC-5 cDNA in stably transfected Chinese hamster ovary (CHO) cells, observed time-dependent activation of Cl
currents
that showed moderate outward rectification, were blocked by DIDS, and
had a selectivity sequence of
I
> Cl
. It is unclear whether
the time-dependent activation of the currents observed by Sakamoto et
al. (24) for rClC-5 expressed in CHO cells is related to the use of a
different expression system. We note that xClC-5 expressed in oocytes
showed features common to mammalian ClC-5 proteins expressed both in
Xenopus oocytes (i.e., strong outward
rectification, time independence) and in CHO cells (i.e., similar
selectivity sequence and DIDS sensitivity).
RPA analysis results showed the presence of significant amounts of
endogenous xClC-5 message in Xenopus
oocytes. Nonetheless, injection of xClC-5 cRNA unequivocally led to a
10- to 20-fold increase of the total oocyte conductance that was due to
the development of an anion pathway. Consequently, it is reasonable to
assume that this current was due to overexpression of xClC-5 protein, although we cannot discern whether xClC-5 is truly a channel of a
channel regulator. Furthermore, we cannot exclude that the observed current was mediated by activation of an endogenous channel due to
overexpression of a structurally unrelated protein (5). Further studies
are needed to assess the single-channel properties of this protein in
other expression systems. In addition, we must note that coexpression
of different members of the ClC family has been shown to produce
Cl currents that are
different from those of the same clones expressed individually (18).
Lorenz and co-workers (18) concluded that this phenomenon was due to
the formation of functional heteroligomeric channels with novel
properties. Given the high homology of several ClC channels between
even poorly related species (for example, 84% homology between xClC-5
and hClC-5), such interactions could also occur between expressed
proteins and endogenous proteins in
Xenopus oocytes. However, these
interactions could affect the expressed
Cl
current only if the
quantity of endogenous protein is sufficient to allow the inclusion of
at least one endogenous protein per heteroligomeric unit.
The role of xClC-5 in epithelial ion transport, and in the maintenance of Vm or cell volume, and the factors that regulate this protein remain to be investigated. Such an attempt was performed by Uchida et al. (28), who reported that the abundance of mRNA for the kidney-specific ClC channel, ClC-K1, was increased in renal cells following dehydration in rats. An advantage of the A6 cell line is that it allows one to precisely control growth conditions. Therefore, one can assess possible regulation of expression of xClC-5 by environmental factors or hormones or during differentiation. In addition, this system may be a valuable model for elucidating whether xClC-5 is expressed in a polarized manner in epithelial cells.
In summary, the A6 cell line expresses a ClC channel that has high homology to hClC-5. The use of cultured A6 epithelial monolayers has advantages that may be useful for understanding the fundamental biological function of ClC-5 proteins and the pathological conditions that result from their malfunction.
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ACKNOWLEDGEMENTS |
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It is a pleasure to thank Dr. Steve King and Dr. Javier Navarro for laboratory facilities and help and also John Embesi and C. Raschi for technical support.
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FOOTNOTES |
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This work was funded by John Sealy Memorial Endowment Grant 2566-94 (N. K. Wills), National Science Foundation Grant IBN-96-29733 (N. K. Wills), North Atlantic Treaty Organization Grant CRG-921221 (J. Ehrenfeld), Centre National de la Recherche Scientifique (France) URA 1855, and the Commissariat à l'Energie Atomique (France).
Current address of S. Lindenthal: Laboratoire Jean Maetz, URA CNRS/CEA 1855, B.P. 68, 06320 Villefranche-sur-Mer, France.
Address for reprint requests: N. K. Wills, Dept. of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555.
Received 21 February 1997; accepted in final form 8 May 1997.
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