Identification of an acid-activated
Cl
channel from human
skeletal muscles
Masanobu
Kawasaki,
Toshiko
Fukuma,
Kazushi
Yamauchi,
Hisato
Sakamoto,
Fumiaki
Marumo, and
Sei
Sasaki
Second Department of Internal Medicine, Tokyo Medical and Dental
University, Tokyo 113-8519, Japan
 |
ABSTRACT |
ClC-4 gene was isolated as a putative
Cl
channel. Due to a lack
of functional expression of ClC-4, its physiological role remains
unknown. We isolated a human ClC-4 clone (hClC-4sk) from human skeletal
muscles and stably transfected it to Chinese hamster ovary cells. Whole
cell patch-clamp studies showed that the hClC-4sk channel was activated
by external acidic pH and inhibited by DIDS. It passed a strong outward
Cl
current with a
permeability sequence of I
> Cl
> F
. The hClC-4sk has
consensus sites for phosphorylation by protein kinase A (PKA); however,
stimulation of PKA had no effect on the currents. hClC-4sk mRNA was
expressed in excitable tissues, such as heart, brain, and skeletal
muscle. These functional characteristics of hClC-4sk provide a clue to
its physiological role in excitable cells.
human ClC-4sk; ClCN4; outwardly
rectifying chloride channel; acidification
 |
INTRODUCTION |
OUTWARDLY RECTIFYING
Cl
channels (ORCC) play
roles in cell volume regulation (8, 27, 40), driving the fluid
secretion from secretory glands (14, 33), and intracellular vesicular acidification (25). Two ORCCs were cloned recently, ClC-3 and ClC-5
(20, 30, 36). They can be classified into a subset of the voltage-gated
Cl
channel superfamily, the
ClC family (1, 5, 11, 13, 17, 35-37, 39). This ORCC subset
consists of three members: ClC-3, ClC-4, and ClC-5, which are ~80%
identical to each other. The outward
Cl
current elicited by
ClC-3 was inhibited by phorbol ester and was stimulated by cell
swelling (9). ClC-3 is thought to be important in cell swelling. The
ClC-5 channel also exhibited outwardly rectifying
Cl
currents (24, 30, 36). A
variety of its gene mutations have been identified in patients with
Dent's disease (24, 31). ClC-5 may be involved in vacuolar
Cl
transport, contributing
to their acidification in renal tubules. In contrast, two
laboratories had already isolated the ClC-4 cDNA clone; however,
both failed to express it functionally (16, 39). It remains
unknown whether the ClC-4 channel is indeed an ORCC as ClC-3 and -5.
In the present study, we isolated ClC-4 (hClC-4sk) cDNA
clone from human skeletal muscle and established stably transfected mammalian cell lines [Chinese hamster ovary (CHO) cells]
expressing hClC-4sk channels. We characterized the expressed hClC-4sk
channel using a patch-clamp technique. We observed outward
Cl
currents that were
activated by extracellular strong acidification.
 |
MATERIALS AND METHODS |
Isolation of human ClC-4 cDNA clones and DNA
sequencing. We performed a PCR cloning strategy using
HIT-T15, a Syrian hamster pancreatic
-cell, as a source of mRNA. One
microgram of HIT-T15 cell total RNA was reverse transcribed at 42°C
for 60 min and then heated at 94°C for 5 min (Avian myeloblastosis
virus RNA-dependent DNA polymerase; Boehringer Mannheim). The
synthesized cDNA was used for subsequent PCR in the following profile:
94°C for 1 min, 55°C for 1 min, 72°C for 3 min, 35 cycles.
PCR primers used were as follows: sense strand
CCGGATCCGGNATHCCNGARHTNAARAC and antisense strand
CCGAATTCRTGNACNARNGGNCCYTCYTT (where N = A/C/G/T; H = A/C/T; R = A/G;
and Y = C/T). The PCR product of expected size (318 bp) was cut with
EcoR I and
BamH I, ligated into
EcoR I and
BamH I cut pSPORT1 (BRL), and then
sequenced. The sequence revealed the existence of a new ClC channel;
however, it was highly homologous to human retina ClC-4 (gene symbol:
ClCN4; 90.1%). Northern blot analysis
using this PCR clone (HIT38) as a probe revealed abundant expression in
rat brain and rat skeletal muscle. Therefore, a human skeletal muscle
library in
gt11was screened for the isolation of a full clone. The
library was screened under high stringency (6× sodium
chloride-sodium phosphate-EDTA, 5× Denhardt's
solution, 1% SDS, 100 µg/ml salmon sperm DNA, and 50% formamide at
42°C) using an HIT 38 insert labeled with
[
-32P]dCTP (3,000 Ci/mmol; Amersham). Two clones with inserts of 3.5 and 4.5 kb were
subcloned into pUC 118, designated as hClC-4sk. Nested deletion clones
were prepared using the Erase-A-Base system (Promega) and were
sequenced using T7 DNA polymerase by the chain termination method or
the dideoxy chain termination method using fluorescence-labeled primers
on an automated sequencer. Antisense strand was sequenced using
synthetic primers.
hClC-4sk-expressing cell line. The
Sal I and blunt-cut fragment of
ClC-4sk was ligated into a mammalian expression vector, pMAM2-BSD.
CHO-K1 cells (obtained from Japanese Collection of Research
Bioresources) were grown in Ham's F-12 Nutrient Mix
(GIBCO) supplemented with 10% FBS at 37°C in 5%
CO2. Conventional intranuclear microinjection of the expression plasmid vector was carried out using
an Eppendorf transjector 5246 and micromanipulator 5171 attached to a
Zeiss inverted phase-contrast microscope (30). Cells containing stably
integrated copies of transfected recombinant plasmid were selected by
adding blastcidin S (BSD) to the growth media at a concentration of 10 µg/ml. After selection for 2 mo, BSD-resistant cell clones were
isolated and transferred to separate culture dishes for expansion and
analysis. Stable expression of the transfected plasmid was confirmed by
Northern blot analysis. Total RNA was isolated from
~107 cells of each BSD-resistant
clone treated with or without 2 µM dexthamethasone for 24 h (22).
Twenty micrograms of each sample of total RNA were resolved in a
formaldehyde-0.7% agarose gel and were blotted onto a nylon membrane.
The membrane was hybridized overnight with
106
counts · min
1 · ml
1
of ClC-4 cDNA probe labeled with
[
-32P]dCTP
(Amersham) by random priming (Promega). A human multiple-tissue Northern blot with 2 µg of
poly(A)+ RNA/tissue
(Clontech) was also hybridized under the same condition. Hybridization
was visualized by autoradiography.
The RT-PCR reaction was performed to distinguish hClC-4
from other Cl
channels. Total RNAs were isolated from each cell of the
ClC-3-expressing cell (C21), hClC-4sk-expressing cell (C53), and
ClC-5-expressing cell (J27). Total RNA (1 µg) was reverse transcribed
at 50°C for 30 min and then heated at 94°C for 5 min (Avian
myeloblastosis virus RNA-dependent DNA polymerase; Boehringer
Mannheim). The synthesized cDNA was used for subsequent PCR in the
following profile: 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, 40 cycles. Specific sense and antisense primers for hClC-4sk
transcript were designed as follows: sense,
5'-ATGGATTTCGTCGATGAGCCGTTCCCTGATGT-3'; antisense,
5'-TAGAAGTCCAGCAACACTGCTCATGGCTATAC-3'. The expected size
of the PCR product is 316 bp for the hClC-4sk transcript. PCR products
were analyzed on a 1.2% agarose gel. To identify the products as a
partial fragment of the hClC-4sk, they were subcloned into pCR 2.1 (Invitrogen) and sequenced using the dideoxy chain termination method
using fluorescence-labeled primers on an automated sequencer (ABI 377).
Electrophysiological characterization.
Conventional patch-clamp techniques were used to record whole cell
currents from ClC-4- or ClC-4 E224A-transfected CHO cells. A mutation
of E224A in ClC-4sk was made using a PCR mutation strategy
(Stratagene). All transfected cells for expression studies were
cultured in a dexamethasone-free medium. The ClC-4sk-transfected cells
treated with dexamethasone produced huge currents and a burst in many
occasions. To obtain stably whole cell currents in broad voltage ranges
and for a long time period, we had to lower the expression level of
hClC-4sk. Currents were recorded at room temperature (20-24°C)
with an EPC-7 patch-clamp amplifier (List-Electronic; West Germany),
and the data were stored on a DAT recorder (DAT-200; Sony, Tokyo,
Japan). Records were sampled at 2,000 points/s and were analyzed on a Compaq ProLinea 4/50 computer using Axon version 6.0 software. The
obtained data were transferred to a Macintosh 550c computer and were
analyzed using Excel 2.2 software. In the whole cell configuration, the
pipette solution contained (in mM): 130 potassium gluconate, 20 KCl, 5 PIPES, 1 EGTA, and 100 µg/ml nystatin (stock solution 25 mg/ml in
DMSO; see Ref. 23), pH 7.2. The bath solution contained (in mM) 130 tetraethylammonium chloride, 1 MgCl2, 10 PIPES, 1 EGTA, and 0.51 CaCl2 (pH 7.20, 6.0, and 4.5). In
the study of anion selectivity, the relative anion permeabilities were
determined on the basis of the relative current after the replacement
of Cl
with other anions
using an SF-77B Perfusion Fast-Step (Warner Instrument). The bath
solution was 120 mM NaF for
F
, 120 mM NaI for
I
, and 120 mM NaCl for
Cl
(pH 4.5). The tonicity
of the standard pipette and bathing medium was measured by
freezing-point depression and was adjusted to 280 mosmol with maltose.
All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless
otherwise noted. Data were filtered at 1 kHz and were sampled at 2 kHz.
 |
RESULTS |
Isolation of hClC-4sk.
Figure 1 shows the primary structure of
hClC-4sk deduced from the nucleotide sequence of the cDNA isolated from
the human skeletal muscle cDNA library. The hClC-4sk
Cl
channel is composed of
760 amino acid residues. The nucleotide sequence of hClC-4sk had seven
nucleotides that were different from that of the
ClCN4 isolated from a retina cDNA
library (39); as a result, it possessed four different amino acids
(residues R178A, Y498I, Y499I, and N659K, see Fig. 1). The
overall amino acid identities to the
ClCN3 and
ClCN5 were high (78% amino acid sequence identity to ClCN3; 78% to
ClCN5). Two potential
N-glycosylation sites are found at
residues 119 and 421. There are four consensus sequences for
phosphorylation sites for protein kinase C at positions Thr43,
Thr50,
Thr362, and
Ser462 and two potential
phosphorylation sites for protein kinase A at positions
Thr362 and
Thr363 (21). Full-length hClC-4sk
cDNA probe hybridized with several bands, mainly at 4.4 and 7.5 kb,
under a high stringent condition in human tissues (Fig.
2A).
The expression was observed to be highest in the skeletal muscle.
Moderate expression was also observed in the heart and the brain. The
7.5-kb transcript could also be detected in the lung, liver, pancreas,
and testis at much lower levels. These results were consistent with
those of van Slengtenhorst et al. (39).

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Fig. 1.
Amino acid sequence of ClC-4 from human skeletal muscle (hClC-4sk).
Different amino acids between ClC-4 gene
(ClCN4) and hClC-4sk are underlined.
, Conserved N-linked glycosylation sites are indicated by filled
diamonds; **, potential phosphorylation sites for protein kinase A. Nucleotide sequence data reported in this paper will appear in the
DDBJ/EMBL/GenBank nucleotide sequence databases with the accession
number AB019432.
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Fig. 2.
A: Northern blot analysis of hClC-4sk
expression in different human tissues.
Poly(A)+ RNA (~2 µg/lane) from
various human tissues were loaded in each lane and subsequently
hybridized with the full-length hClC-4sk cDNA probe. Markers of
transcript size (in kb) are indicated.
B: stable expression of hClC-4sk.
Northern blot analysis of transfected cell lines for expression of
hClC-4sk (top). Every lane on an
agarose gel contained 10 µg of total RNA from each transfected cell
line. +, Pretreatment with 2 mM dexamethasone (Dex) for 1 day; ,
without pretreatment. Lane 1, clone 11 Dex ( ); lane 2, clone 53 Dex
( ); lane 3, clone 11 Dex (+);
lane 4, clone 53 Dex (+). Total RNAs
transferred to the nylon membrane were stained by ethidium bromide and
visualized by ultraviolet light
(bottom).
C: expression of hClC-4sk without
dexamethasone detected by RT-PCR. hClC-4sk PCR products could be
obtained from the hClC-4-transfected cells without dexamethasone.
RT-PCR for hClC-4sk at 40 cycles revealed undetectable level of gene
expression from ClC-3- and ClC-5-transfected cells.
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Functional expression of hClC-4sk in CHO
cells. CHO-K1 were stably transfected with the coding
sequence of cloned hClC-4sk using an expression vector pMAM2-BSD. After
the selection with BSD for 2 mo, 85 clonal cell lines derived from the
transfected cells were examined for hClC-4sk mRNA expression, and two
clonal cell lines (clone 11 and clone 53) were selected by Northern
blot analysis (Fig. 2B). We did
RT-PCR to reveal a level of gene expression of hClC-4sk from
ClC-4-transfected cells in the absence of dexamethasone (Fig.
2C). As negative controls, we used
ClC-3- and ClC-5-transfected cells to show the primer set specific for
hClC-4sk. To identify the products as a partial fragment of the
hClC-4sk, they were subcloned and sequenced (4/4).
To examine the expression of hClC-4sk channels on the cell surface, we
assayed the transfected cells by the patch-clamp technique in the whole
cell configuration. In a physiological condition (pH 7.2, 280 mosmol),
ClC-4sk-transfected cells showed a very small current (165 ± 33.8 pA; mean ± SE at +100 mV membrane potential, n = 10). Nontransfected (134 ± 61 pA, n = 9) and
vector-alone-transfected (167 ± 53 pA,
n = 6) CHO-K1 cells produced a similar
level of current amplitude (Fig. 3). In the
previous reports of other laboratories, ClCN4 channels were not functionally
expressed in Xenopus oocytes and
mammalian cells (16, 39). The present result at pH 7.2 was consistent
with those previous reports.

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Fig. 3.
Functional expression of hClC-4sk. Nontransfected pH 7.2, representative trace of the nontransfected Chinese hamster ovary (CHO)
cells on exposure to a standard bath solution of pH 7.2; nontransfected
pH 4.5, exposure to the pH 4.5 bath solution; vector alone pH 7.2, representative trace of mocked CHO cells on exposure to a standard bath
solution of pH 7.2; vector alone pH 4.5, exposure to the pH 4.5 bath
solution; ClC-4 pH 7.2, representative trace of the
hClC-4sk-transfected CHO cells on exposure to the pH 7.2 bath solution;
ClC-4 pH 4.5, exposure to the pH 4.5 bath solution; ClC-4 pH 6.0, exposure to the pH 6.0 bath solution. The holding potential was changed
from 100 to +100 mV stepped by +20 mV. Bottom
right: current-voltage relationships of hClC-4sk
channel in low pH (pH 4.5 or 6.0). Whole cell currents were measured
between 100 and +100 mV. Ensemble averages of hClC-4sk channel
currents were constructed for each of the data points plotted
(n = 6).
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Recently, an immunohistological study demonstrated that the ClC-5
channel protein was colocalized with
H+-ATPase at intracellular
vesicles in renal proximal tubule (15). Their localization suggested
that the ClC-5 channel probably plays a role in vesicular
acidification. Intravacuolar environment is maintained at pH of
4.5-5.0 in mammalian cells (28). It would be possible that ClC-3,
ClC-4, and ClC-5 are regulated by extracellular pH
(pHo). We examined the effect of
acidification on the hClC-4sk-expressing cells. When pH of the bath
solution was changed from pH 7.2 to 4.5, an outwardly rectifying
current appeared in hClC-4sk-transfected cells (1,383 ± 187 pA;
n = 10/10), whereas no significant
current appeared in nontransfected (134 ± 34 pA;
n = 7) and vector-alone-transfected (215 ± 108 pA, n = 6) cells (Fig.
3). The acidic pH-induced currents were activated instantaneously by
depolarizing voltage with further slow activation. Stepping back to the
holding potential caused deactivation instantaneously. The predicted
reversal potential of the current activated by acidic solution was near
the equilibrium potential for
Cl
, suggesting that the
current was carried mainly by
Cl
. Steady-state
current-voltage curves of activated channel currents revealed a strong
outward rectification (Fig. 3, bottom
right) that is stronger than that of ClC-3 and -5 in
CHO cells (19, 30). When hClC-4sk-expressing cells were exposed to pH
6.0, hClC-4sk channels were activated by this
pHo in some cells
(n = 5/9), and current amplitudes were
about one-half that of pHo 4.5 (682 ± 176 pA; Fig. 3). In the remaining inactivated cells (4/9),
reducing bath pH further to <5.0, the channels were activated (n = 4/4). When increasing the
pHo value above 6.5, the ClC-4 was
always inactive.
Relative anion selectivity to
Cl
,
I
, and
F
was compared (Fig.
4). In the whole cell currents, solutions
containing iodides gave current amplitudes 1.42 ± 0.36 (at 100 mV;
n = 4) times larger than those with
Cl
. The current reversed at
more negative potentials when
Cl
was replaced by
equimolar I
.
F
was less permeable than
Cl
(0.26 ± 0.05).
Replacement of extracellular
Cl
with
F
shifted the reversal
potential to more positive values. The anion selectivity sequence of
hClC-4sk currents was I
> Cl
> F
(7). The anion
permeability sequence of the hClC-4sk was identical to that of the rat
ClC-3 and rat ClC-5 (18, 25, 30). Extracellular 1 mM DIDS, an inhibitor
of Cl
channels, decreased
the conductance by 77.5 ± 4.6% (n = 3). Thus the ClC-4 channel is a DIDS-sensitive ORCC.

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Fig. 4.
Anion permeability sequence of hClC-4sk. Representative trace series of
hClC-4sk whole cell currents in the different anion replacement medium.
Cl was totally replaced
with the same concentration of the anion
(I ,
Cl , and
F ) at pH 4.5. Cells were
subjected to the pulse protocol as described in Fig. 3.
A: NaF;
B: NaI;
C: NaCl; and
D: relative current-voltage
relationships of hClC-4sk in each anion. Currents at given test
potentials and ion concentrations were normalized to the current at
+100 mV in NaCl buffer for individual cells. Ensemble averages of
hClC-4sk channel currents were constructed for each of the data points
plotted (n = 4).
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Figure 5 showed representative sequential
traces of hClC-4sk current regulated by
pHo. In nontransfected cells, when
pH of the bathing solution was changed from pH 7.2 to 4.5 using the triple-barreled method, endogenous
Cl
currents evoked by pulse
stimuli remained very small. In contrast, on exposure to the bath
solution of pH 4.5, an outwardly rectifying Cl
current appeared from
the hClC-4sk-expressing cell within a few minutes [mean time to
half-maximal activation = 79.2 ± 6.8 (SD) s,
n = 6; Fig.
5B,
top]. Treatment of pH 8.0 bath
solution sharply decreased the currents (Fig.
5B,
middle), and this inhibitory effect
quickly disappeared after changing the bath solution from pH 8.0 to 4.5 (mean time to half-maximal activation = 11.6 ± 5.8 s,
n = 6), indicating that the underlying
mechanisms for the initial and second activation by acid pH may be
different. Next, we tested the effects of activators of protein kinase
A on a low pH-induced Cl
current. 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic
monophosphate (200 µM) with 1 mM IBMX and 10 µM forskolin at pH 4.5 had no significant effect on the current (Fig.
5B,
bottom), suggesting that the
pH-sensitive hClC-4sk channel was not regulated by protein kinase A. To
confirm that the Cl
currents observed in hClC-4sk-transfected cells indeed passed through
the hClC-4sk channels, we introduced a point muta tion, E224A, to
hClC-4sk cDNA and characterized the functional properties of hClC-4sk
E224A. The E224A mutant-transfected cells generated inwardly instead of
outwardly rectifying Cl
currents, even at pHo 7.2 (n = 3, Fig.
6).

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Fig. 5.
Effect of pH on the hClC-4sk channel currents.
A: nontransfected;
B: ClC-4. Representative sequential
traces of whole cell currents from hClC-4sk-transfected cells
sequentially exposed to pH 7.2, 4.5, and 8.0. Labeled arrows indicate
changes of bath solution pH. "Forskolin" indicates the
application of forskolin mixture. Holding potential was clamped at
±50 mV.
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Fig. 6.
Mutant expression of hClC-4sk E224A.
A: representative trace of the
hClC-4sk E224A-transfected CHO cells on exposure to the pH 7.2 bath
solution. Holding potential was changed from 100 to +100 mV
stepped by +20 mV. B: current-voltage
relationships of hClC-4sk E224A channel. Whole cell currents measured
between 100 and +100 mV. Ensemble averages of hClC-4sk channel
currents were constructed for each of the data points plotted
(n = 3).
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 |
DISCUSSION |
The ClCN4 gene was isolated from the
Xp22.3 region using a positional cloning strategy (39). A deletion of
the Xp22.3 region caused a psychomotor delay and mental retardation in
the patient (3). The ClCN4 gene
possibly contributes to the pathogenesis of these neuronal disorders.
In the present study, human ClC-4 cDNA was isolated from a human
skeletal muscle cDNA library by a sequence homology-based strategy. We
successfully obtained stable functional expression in CHO cells.
The present nucleotide sequence of hClC-4sk showed seven nucleotides in
the ORF-coding sequence different from that of
ClCN4. These nucleotide mutations
changed four amino acids of the ClCN4 amino acid sequence. However, the amino acids at these four positions of hClC-4sk were conserved in those of rat and mouse ClC-4 and human
genomic DNA. The human retina cDNA alone is different. Probably, its
nucleotide sequence differences are caused by sequencing or cloning errors.
In this study, the hClC-4sk channel was activated by extracellular
acidic pH. On exposure to acidic bath solutions, hClC-4sk generates a
very strong outwardly rectifying
Cl
current (Fig. 3).
Mock-transfected CHO cells did not produce Cl
currents in the acidic
bath solutions. The E224A mutation altered its channel rectification
from strongly outward to inward (Fig. 6). These data proved that
hClC-4sk actually generated
Cl
currents. These
properties of the ClC-4 whole cell currents, namely strong outward
rectification, DIDS sensitivity, anion conductivity, and insensibility
to protein kinase A, were consistent with the previous reports of ClC-3
and ClC-5, except for activation by acidification (9, 19, 20,
30). Thus the ClC-4 channel is an actual member of the
ORCC subfamily.
If we assume that ClC-4 is a vacuolar
Cl
channel, the expected
orientation of the hClC-4sk channel within a vacuolar membrane would
make it better suited for
Cl
efflux rather than
Cl
influx for vacuole
acidification. Thus it is necessary for the ClC-4 to pass through
influx of Cl
. However, the
hClC-4sk inward current was inactive in the present study. Native
vesicular Cl
conductance
was regulated by protein kinase A (2, 26). hClC-4sk was not regulated
by protein kinase A (Fig. 5) like other members of the ORCC subfamily
(ClC-3 and ClC-5; see Refs. 20 and 30). It would be possible that a
regulatory factor(s) is lacking to obtain inward currents and protein
kinase A activation properties in CHO cells. With regulator(s), the
hClC-4sk channel may play a role of vacuolar acidification in excitable cells.
pHo can modulate the channel
activity of some Cl
channels. Acidic pHo diminished
time-dependent inactivation of ClC-1 inward currents; thus, the
steady-state component was enhanced (29). Hyperpolarization-activated
ClC-2 currents were stimulated by acidic
pHo (32, 34). Reducing
pHo from 7.3 to 6.5 decreased inwardly rectifying Cl
currents from the epithelial cells of the choroid plexus (18). These
reports showed that pHo modulated
channel activity. During the revision of this draft, an expression
study of ClC-4 was published by Friedrich et al. (12). Interestingly,
they showed that the ClC-4 channel was inhibited by external low pH and
has an anion permeability sequence of
Cl
> I
. In contrast, the
hClC-4sk channel was activated by external low pH in an all-or-none
fashion. It has an anion conductivity sequence of
I
> Cl
. The reason for the
functional discrepancies between their results and ours is not clear.
The expression system (Xenopus oocyte
vs. CHO-K1 cells) and experimental condition (perforated vs.
cell-attached whole cell current) are different. It is possible that
the ClC-4 channel is modulated by endogenous intracellular factors.
The time course of the initial activation of hClC-4sk
by acidic pH was always longer than that of the second activation. When extracellular acid solution was first exposed to the
hClC-4sk-expressing cells, they always generated
Cl
currents after a few
minutes. Alkaline
pHo of 8.0 quickly shut down the
outwardly rectifying Cl
current, which had already been activated by acidic
pHo (Fig. 5). Blockade of hClC-4sk
by an increase in pHo was again
quickly recovered by a low pHo.
The slow initial activation and the fast pH regulation afterward are
possibly caused by different regulatory mechanisms. The pH gradient
along the exocytotic pathway may be important for the sorting proteins
and vesicle trafficking (6, 25). We speculate that the ClC-4 channels
might localize in organelles at neutral pH. Decreasing
pHo may activate the exocytotic pathway, allowing the sorting of vesicles containing the ClC-4 channels
into the plasma membrane. A number of the hClC-4sk channels increased,
and it produced Cl
currents. Once hClC-4sk channels were put on the plasma membrane, they
should be quickly regulated by
pHo, suggesting that the ClC-4 channel protein may be directly modified by extracellular acidification through possible protonation of an external amino acid residue.
 |
ACKNOWLEDGEMENTS |
We thank K. Hayashi for help with cloning of hClC-4sk.
 |
FOOTNOTES |
This work was supported by grants-in-aid from the Ministry of
Education, Science, and Culture, Japan.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Kawasaki, Second Dept. of Internal Medicine, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519,
Japan.
Received 4 January 1999; accepted in final form 17 August 1999.
 |
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