From the Zentrum für Molekulare Neurobiologie Hamburg (ZMNH),
Hamburg University, Martinistrae 52, D-20246, Hamburg, Germany
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ABSTRACT |
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ClC-4 and ClC-5, together with ClC-3, form a
distinct branch of the CLC chloride channel family. Although ClC-5 was
shown to be mainly expressed in endocytotic vesicles, expression of ClC-5 in Xenopus oocytes elicited chloride currents. We now
show that ClC-4 also gives rise to strongly outwardly rectifying anion currents when expressed in oocytes. They closely resemble ClC-5 currents with which they share a NO3 The CLC1 family of
chloride channels, originally defined by the ClC-0 chloride channel
from Torpedo electric organ (1), comprises nine known
members in mammals (2). Mutations in three of the corresponding genes
are known to cause human disease: mutations in ClC-1 cause myotonia (3,
4), mutations in ClC-Kb Bartter's syndrome (5), and mutations in ClC-5
cause Dent's disease (6).
Dent's disease is an X chromosome-linked disorder and has two main
symptoms: hypercalciuria, which leads to kidney stones, nephrocalcinosis, and renal failure, and second, low molecular weight
proteinuria (7). The proteinuria points to a defect in endocytosis of
proximal tubular cells. Indeed, ClC-5 is expressed in the proximal
tubule and in intercalated cells of the distal nephron (8). Both cell
types are involved in endocytosis. The localization of ClC-5 in
intracellular vesicles and its co-localization with the proton pump
suggests that it provides an electrical shunt necessary for an
efficient acidification of these vesicles. This defect in
intravesicular acidification probably leads to the impaired endocytosis
of proteins observed in Dent's disease. In transfected cells, ClC-5
was present in intracellular vesicles. In addition, there was also some
labeling of the plasma membrane (8).
Consistent with a plasma membrane localization, expression of ClC-5 in
Xenopus oocytes elicited chloride currents (9). These had a
NO3 Despite these open questions, ClC-5 is the best understood member of
the ClC-3/4/5 branch of the CLC gene family. These three proteins are about 80% identical (9). In previous studies, we did not
observe currents upon ClC-4 expression (14). While some groups reported
that ClC-3 expression gave no currents (14, 15), others reported
outwardly rectifying currents with an I The present study has two major aims. First, to compare several
electrophysiological properties of currents elicited by these highly
homologous proteins. Second, to prove that these currents are directly
due to these gene products by introducing mutations that change their
properties. For the first time, ClC-4 gave chloride currents when
expressed in Xenopus oocytes or HEK293 cells. These resemble
ClC-5 currents in many respects, but differ slightly in voltage
dependence and pH sensitivity. Using various point mutations we
demonstrate that these currents are indeed mediated by ClC-4 and ClC-5.
Surprisingly, their properties differ significantly from those reported
for ClC-3 (16-19), and we could not detect currents upon ClC-3 expression.
ClC-4 and ClC-5 Constructs--
A human ClC-4 cDNA was
cloned into pTLN (23) or pCIneo (Promega, Madison, WI). It differed
from the published sequence (24) (GenBankTM accession
number X77197) at the following residues: Ala instead of Arg
(GenBankTM) at 178; Ile-Ile instead of Tyr-Tyr at 498 and
499; and Lys instead of Asn at 659. The residues we found are also
present in the rat (GenBankTM accession number Z36944) and
mouse (GenBankTM accession number S47327) cDNAs. The 5'
end of ClC-4 contains three ATGs in frame (at amino acid numbers 1, 7, and 14; the last one corresponds to the ClC-5 initiator ATG). We could
not detect functional differences between these and therefore used the
first ATG for all subsequent studies. Amino acids are numbered starting from this methionine. Mutations were introduced by recombinant polymerase chain reaction. All polymerase chain reaction-derived fragments were fully sequenced. The cDNA of ClC-5 contained the rat
sequence between the initiator ATG and the DraIII
restriction site, which is 100% identical to the human sequence on the
protein level.
Expression in Xenopus laevis Oocytes and Voltage-Clamp
Studies--
Using SP6 RNA polymerase capped cRNA was transcribed from
the constructs after linearization. 10-25 ng of cRNA were injected into Xenopus oocytes isolated by manual defolliculation as
described (1). Oocytes were kept at 17 °C in modified Barth's
solution (88 mM NaCl, 1.0 mM KCl, 1.0 mM CaCl2, 0.33 mM
Ca(NO3)2, 0.82 mM
MgSO4, 10 mM HEPES, pH 7.6). Two-electrode
voltage-clamp measurements were performed at room temperature 2-3 days
after injection using Turbotec 05 or 10C amplifiers (NPI Instruments,
Tamm, Germany) and pClamp 5.5 software (Axon Instruments). Currents
were recorded in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM Na-HEPES, pH 7.4).
For anion replacement, 80 mM Cl Whole Cell Patch-Clamp Measurements--
HEK293 cells were
transiently transfected with ClC-4 or ClC-5 cDNA (WT or mutants,
subcloned into pCIneo) using LipofectAMINETM (Life
Technologies, Inc.) according to the manufacturer's procedures. To
identify transfectants, a green fluorescent protein construct (pEGFP;
CLONTECH) was co-transfected. Whole cell
patch-clamp measurements were performed 30-72 h after transfection at
room temperature in an extracellular solution containing 140 mM NMDG, 2 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.4, using
an Axopatch 200A amplifier (Axon Instruments) and pClamp5.5 software.
Patch pipettes were pulled from borosilicate glass to 2-5 µm tip
diameter and filled with either high-Cl intracellular solution (140 mM NMDG, 2 mM MgCl2, 2 mM EGTA, 5 mM HEPES, pH 7.4) or low-Cl
intracellular solution (120 mM NMDG-aspartate, 20 mM NMDG-Cl, 2 mM MgCl2, 2 mM EGTA, 5 mM HEPES pH 7.4). Pipette
resistances were in the range of 1-5 M To reinvestigate the functional expression of ClC-4, we cloned the
human ClC-4 cDNA into an optimized expression vector (23) and
injected derived cRNA into Xenopus oocytes. After 2-3 days, two-electrode voltage-clamping revealed strongly outwardly rectifying chloride currents (Fig. 1a)
that were absent from control oocytes. Currents activated rapidly at
positive voltages and in many respects resemble ClC-5 currents (9). No
tail currents could be detected when stepping back to negative
voltages. Similar currents were observed when ClC-4 or ClC-5 were
studied in transfected HEK293 cells (Fig. 1, c and
d, respectively). This excludes that these currents are due
to the activation of a chloride channel specific for Xenopus
oocytes. Since the intracellular solution is buffered with EGTA, both
channels do not depend on intracellular calcium. In contrast to ClC-5,
where currents begin to activate at voltages more positive than +20 mV
((9) and Fig. 1e), ClC-4-induced currents are already
visible at slightly more negative potentials and depend less steeply on
voltage (Fig. 1e). Partial replacement of extracellular chloride by other anions indicated a
NO3 > Cl
> Br
> I
conductance
sequence that differs from that reported for the highly homologous
ClC-3. Both ClC-4 and ClC-5 currents are reduced by lowering
extracellular pH. We could measure similar currents after expressing
either channel in HEK293 cells. To demonstrate that these currents are
directly mediated by the channel proteins, we introduced several point
mutations that change channel characteristics. In ClC-5, several point
mutations alter the kinetics of activation but leave macroscopic
rectification and ion selectivity unchanged. A mutation (N565K)
equivalent to a mutation reported to have profound effects on ClC-3
does not have similar effects on ClC-5. Moreover, a mutation at the end
of D2 (S168T in ClC-5) changes ion selectivity, and a mutation at the
end of D3 (E211A in ClC-5 and E224A in ClC-4) changes voltage
dependence and ion selectivity. This shows that ClC-4 and ClC-5 can
directly mediate plasma membrane currents.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
> Cl
> Br
> I
conductance sequence and were
strongly outwardly rectifying. Currents were detectable only at
voltages more positive than +20 mV. When we analyzed ClC-5 mutations
found in Dent's disease (6, 10-12), currents were either
significantly decreased or abolished. In contrast to ClC-1 mutations in
myotonia (13), we never found changes in rectification, voltage
dependence, or ion selectivity. Thus, we could not rule out the
possibility that these currents were mediated by a different channel
that was activated by expressing ClC-5. Mutations compromising ClC-5
function would then change those currents quantitatively without
changing their characteristics. Given that ClC-5 plays a role in
endocytosis (8), ClC-5 expression may change the plasma membrane
localization of channels that are endogenous to the expression system.
Thus, identifying mutations that change current characteristics seemed
of high priority.
> Cl
selectivity (16-19). The work of Duan et
al. (18) suggests that ClC-3 represents the ubiquitous
swelling-activated chloride channel. These authors introduced a
mutation into ClC-3 that was equivalent to a mutation changing pore
properties in other CLC channels (20-22) and found the expected
changes in ClC-3-induced currents (18).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
was
substituted by equivalent amounts of Br
,
I
, NO3
, or
glutamate. When using different pH values, 5 mM HEPES (for pH 7.4) were replaced by 5 mM MES (for pH 6.5 and 5.5) or 5 mM TAPS (for pH 8.5). Permeability coefficients were
calculated from reversal potential measurements under biionic
conditions using the Goldman-Hodgkin-Katz equation (25).
. Data were usually digitized
at 2 kHz frequency after filtering at 1 kHz.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
> Cl
> Br
> I
conductance sequence (Fig.
1b). This is again similar to ClC-5 (9) and readily
distinguishes these currents from outwardly rectifying, endogenous
oocyte currents that display an I
> Cl
conductance (9). It is not possible to determine permeability ratios as
both channels do not mediate large enough currents at the chloride
equilibrium potential.
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Fig. 1.
Basic electrophysiological properties of
human ClC-4 and ClC-5. a, voltage-clamp traces of ClC-4
expressed in Xenopus oocytes. Currents were measured in
ND96. From a holding potential of 35 mV, the oocyte was clamped in
20-mV steps to voltages between +80 and
100 mV, followed by a
constant test pulse at
80 mV. b, ion selectivity of ClC-4
expressed in Xenopus oocytes and measured as in
a. 80 mM extracellular chloride was substituted
by different anions. (
, ND96;
, 80 mM
I
;
, 80 mM Br
;
, 80 mM NO3
). Data were
averaged from 29 oocytes from 4 different batches (standard errors were
smaller than symbol size). Before averaging, currents of individual
oocytes were normalized to the current at +80 mV in ND96 solution.
c and d, whole-cell patch-clamp traces of HEK293
cells transiently transfected with ClC-4 (c) or ClC-5
(d). Patch pipettes contained the high chloride
intracellular solution, and cells were bathed in the extracellular
solution. From a holding potential of
30 mV, cells were clamped to
voltages between +100 and
100 mV in 20-mV steps, followed by a
constant pulse to
60 mV. e, steady-state I
V
relationships of ClC-4 (
) and ClC-5 (
) currents from whole-cell
experiments in c and d. Amplitudes were taken
from currents at the end of the test pulse and normalized for each
experiment to the value at +80 mV to allow a direct comparison of
channel rectification.
Currents mediated by either ClC-4 or ClC-5 are markedly reduced by extracellular acidification (Fig. 2). A similar pH sensitivity was recently described for a Xenopus CLC channel that may be the species homolog of ClC-5 (26). ClC-5 currents respond to pH changes already in the neutral range and are reduced to less than 50% at pH 5.5 (Fig. 2a). By contrast, ClC-4 currents are nearly unaffected in the neutral pH range, and currents begin to decrease when extracellular pH drops below pH 6.5 (Fig. 2b). We could not measure currents at more acidic pH reliably because currents in Xenopus oocytes became unstable. Nonetheless, our results suggest that the pH dependence of ClC-4 is shifted toward more acidic pH values when compared with ClC-5 (Fig. 2c).
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Although the rather subtle differences between the currents induced by ClC-4 and ClC-5 suggest that they are directly mediated by these proteins, we sought more definitive evidence from mutational analysis. Mutations associated with Dent's disease either abolished or reduced ClC-5 currents without changing their biophysical properties (6, 10-12). S244L and S520P are mutations that resulted in reduced but otherwise typical currents (6). The mutated amino acids are located in putative transmembrane domains D5 and D11, respectively (Fig. 3f). We mutated these critical residues to other amino acids (alanine and threonine). While S244A currents were similar to WT (Fig. 3a), the other mutations significantly slowed the activation of ClC-5 (Fig. 3, b, c, and d). This was observed both in transfected HEK293 cells (Fig. 3) and in Xenopus oocytes (not shown). However, we did not find changes in ion selectivity or in steady-state rectification. As with WT channels, we could not use tail currents to measure the rectification of the open pore.
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Residues affecting ion selectivity and rectification were found in
several regions of CLC channels (27). The first mutation shown to
affect pore properties was K519Q (and K519E) in ClC-0 (20). This lysine
at the end of a large transmembrane block is conserved within ClC-0,
-1, -2, and the two ClC-K isoforms. Both mutations reduced the
Cl > I
selectivity of ClC-0, introduced an
outward rectification, slowed gating, and reduced its single channel
conductance (20, 27, 28). In ClC-3, -4, and -5, an asparagine is
present at the equivalent position. We changed this asparagine to a
lysine in ClC-5 (N565K). Its effect may be opposite to that of K519Q,
particularly since the effect of that ClC-0 mutation may be due to the
charge of the side chain (28). This would predict a more linear I
V relationship and an increase in the
Cl
/I
conductance ratio. However, this is
not the case. While the N565K mutation slightly slowed the activation
of ClC-5 currents (Fig. 3e), it did not change the overall
rectification (Figs. 3e and 4a). It slightly
increased the relative iodide conductance (Fig. 4a). Thus, it is not a mirror
image of the Torpedo K519Q mutation. Nevertheless, it shows
that these currents are directly mediated by ClC-5 and that residues at
the end of D12 influence pore properties.
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Several other regions modulate CLC pore properties. In ClC-0, a conservative mutation (S123T) in a highly conserved stretch (GSGIPE) at the end of D2 increases bromide and iodide conductances relative to chloride (27). The equivalent mutation (S168T) also reduces the ion selectivity of ClC-5 (Fig. 4b). At positive voltages, bromide and iodide conductances are increased with respect to chloride, an effect that is more drastic than that of the ClC-0 S123T mutation (27).
Several studies (19, 29-31) reported that mutations in another highly
conserved region, GKEGP at the end of D3, also have effects on gating
and pore properties. We mutated the glutamate to alanine in ClC-4
(E224A) and in ClC-5 (E211A). There was a drastic effect on gating
(Fig. 4, c and d). Both channels now mediate
significant currents in the negative voltage range. We could not detect
significant current relaxations, suggesting that gating is now either
substantially faster or does no longer depend on voltage. There was
also a slight increase in bromide conductance at positive voltages, the
only range where a comparison to WT channels is possible (Fig. 4,
e and f). The changed voltage dependence allowed
us to measure permeability ratios that indicated a
NO3 (1.5) = Br
(1.45) > Cl
(1.0) = I
ratio for ClC-4(E224A), and a
Br
(1.2) > Cl
(1.0) = NO3
> I
(0.7) sequence
for ClC-5(E211A). It is unclear how these differ from the WT
permeabilities which cannot be measured.
Given the strong similarity between ClC-4 and ClC-5 currents, it is
surprising that ClC-3 was reported to elicit less strongly rectifying
channels with an I > Cl
selectivity
(16-19). In previous experiments, we (9, 14) and others (15) were
unable to obtain ClC-3 currents in Xenopus oocytes. We have
now repeated these experiments with human and guinea pig (18) ClC-3
cDNAs cloned into the expression vectors used in this study. We
again failed to observe currents with human ClC-3 in Xenopus
oocytes. Attempts to express chloride currents with the guinea pig
ClC-3 in transiently transfected HEK293 or NIH3T3 cells were
unsuccessful as well.
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DISCUSSION |
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ClC-4 and ClC-5, together with ClC-3, form a distinct branch of the CLC gene family (2). This branch has received much attention because ClC-5 mutations cause Dent's disease (6) and because ClC-3 was recently proposed (18) to represent the long sought swelling-activated chloride channel. ClC-4 did not elicit currents in previous studies, and attempts to express ClC-3 gave conflicting results (9, 15-19). Furthermore, a formal proof that the strongly outwardly rectifying currents provoked by ClC-5 are directly mediated by this channel was missing.
We now demonstrate that both ClC-4 and ClC-5 directly mediate plasma
membrane currents that are very similar. This includes their extreme
outward rectification, the lack of significant tail currents, and the
NO3 > Cl
> Br
> I
conductance sequence at strongly
depolarizing potentials. Both ClC-4 and ClC-5 currents were sensitive
to extracellular pH. However, they are not identical. Given these
differences, it is unlikely that both proteins activate an identical
endogenous oocyte channel, an otherwise frequently observed phenomenon
with Xenopus oocytes (32). This is also unlikely in view of
the very similar currents in different expression systems
(Xenopus oocytes and HEK293 cells).
The observed pH dependence of ClC-4 and ClC-5 possibly points to an important physiological role of these channels. ClC-5 is expressed in the endocytotic pathway where it may provide the electrical shunt necessary for an efficient acidification of the vesicle interior (8). A similar role in intracellular organelles was proposed for the yeast CLC channel (33-35). Topologically, the vesicle interior corresponds to the extracellular space. An inhibition of chloride currents by acidic intravesicular pH will provide a negative feedback on proton pumping and could be important for setting the pH of these vesicles. Along the endocytotic pathway, the intravesicular pH gets progressively more acidic from early (pH 6.0-6.5) to late (pH 5.0-6.0) compartments (36). The pH dependence of ClC-5 fits well with a localization in the early endocytotic pathway (8). An assignment for the physiological role of the pH dependence of ClC-4 is not yet possible and requires an exact localization of that channel.
It is puzzling that both channels yield significant currents only
at very positive voltages. The voltage across intracellular vesicles is
not well known, but the electrogenic proton pump is expected to create
an inside positive voltage. Topologically this corresponds to a
negative voltage in our measurements, which would prevent a significant
opening of ClC-4 or ClC-5. However, we cannot exclude small currents
due to non-zero open probability at negative voltages. Transport rates
of active pumps are orders of magnitudes lower than those of ion
channels. Hence, even such small channel-mediated currents may suffice
to allow efficient proton pumping. Alternatively, there may be other
subunits that alter the voltage dependence. Some CLC proteins can form
heteromeric channels with novel properties (23), but we observed no
qualitative changes when co-expressing ClC-5 with either ClC-4 or ClC-3
(not shown). CLC channels may also have as yet unknown
-type subunits.
The most compelling evidence for a direct channel function of ClC-4 and
ClC-5 comes from our mutagenesis experiments. Several conservative
amino acid exchanges slowed the activation of ClC-5 currents in both
expression systems. However, changing gating kinetics by mutagenesis
does not prove that these proteins are directly ion channels. Indeed,
gating kinetics is often drastically affected by -subunits that do
not form the pore (37) and may even be changed by expression levels
(38). More convincing than mutations changing kinetics are those that
change pore properties such as ion selectivity and/or rectification.
The pore of CLC channels has not yet been defined. Point mutations at different positions change pore properties in ClC-0, -1, and -2. This includes residues at the ends of transmembrane domains D2 (27), D3 (19, 29-31), and D12 (20-22, 27, 28, 39). Mutations in all three regions also changed properties of ClC-5. A mutation at the end of D12 (ClC-5(N565K)) slightly altered both gating kinetics and conductance ratios. The most drastic effect on conductance ratios was exerted by a mutation in the highly conserved GSGIPE region at the end of D2. A mutation at the end of D3 (ClC-4(E224A) and ClC-5(E211A)) had drastic effects on rectification of both channels and also slightly changed their conductance sequences. Several mutations in the same region in ClC-1 yielded channels with large inward currents (19), as did a mutation in the corresponding region of ClC-0 (40). Many mutations altering the ion selectivity also changed the kinetics or the voltage dependence of gating or both. This has been observed previously also with ClC-0 and ClC-1 (19-21, 27). Indeed, permeation and gating is intimately linked in CLC channels (20, 21, 41, 42). In contrast to ClC-0 (20) and ClC-1 (21), however, anion substitution did not appreciably change the voltage dependence of ClC-4 or ClC-5 (Fig. 1b and Ref. 9). This is compatible with the notion that the conductance ratios of our measurements reflect pore properties and are not just a consequence of changes in open probability.
The Cl > I
selectivity sequence of ClC-4
and ClC-5 agrees well with that of ClC-0 (20), ClC-1 (19, 29), and
ClC-2 (22, 43, 44) and may be a general property of CLC channels. Our data on ClC-5 (9) differ from those of Sakamoto et al. (45). They proposed an I
> Cl
selectivity,
although the shift of reversal potentials upon ion substitutions (45)
suggests a Cl
> I
selectivity. Their
currents are less rectifying and sensitive to the chloride channel
inhibitor DIDS, which was ineffective in our study (9).
Xenopus ClC-5 also elicited less rectifying, DIDS-sensitive
currents with an I
> Cl
selectivity (46).
However, when expressed from vectors optimizing expression in oocytes,
the same authors reported currents similar to those obtained in our
laboratory, and the previously described currents (46) were probably
endogenous to oocytes2 (26).
Indeed, injection into Xenopus oocytes of cRNAs encoding several unrelated proteins can elicit similar outwardly rectifying chloride currents with an I
> Cl
selectivity (41).
Currents mediated by ClC-4 and ClC-5 are very similar but differ
significantly from those reported for ClC-3 (16-19). This is
surprising because these proteins are about 80% identical, and ClC-3
is even slightly more homologous to ClC-4 than is ClC-5. Expression of
ClC-3 in Xenopus oocytes (16) or transfected mammalian cells
(17-19) was associated with outwardly rectifying currents with an
I > Cl
selectivity. However, the channels
reported by Duan et al. (18) and Kawasaki et al.
(17) differ in single channel conductance, rectification, and calcium
sensitivity. Duan et al. (18) suggest that ClC-3 represents
the important swelling-activated chloride channel. This was supported
by a mutation introducing a positive charge at the end of D12 (N579K).
The current-voltage relationship apparently changed from outwardly
rectifying to linear and ion selectivity from
I
> Cl
to Cl
> I
(18). This was expected since a mutation in ClC-0
(K519Q), which neutralizes a charge at this position, has roughly the
opposite effect on selectivity and rectification (20). Surprisingly, the equivalent mutation (N565K) in the highly homologous ClC-5 channel,
which has a Cl
> I
selectivity to begin
with, rather slightly increases iodide conductance relative to chloride
and has no effect on rectification (Fig. 4a). Moreover, we
were again unable to measure ClC-3 currents in oocytes and in
transfected cells. We have no explanation for these discrepancies.
In view of the predominantly intracellular localization of ClC-5 (8),
future studies should address the subcellular distribution of the
highly homologous ClC-3 and ClC-4 channels. Preliminary experiments
show that ClC-4 is also predominantly present in intracellular vesicles.3 It will be
interesting to see whether the (partial) surface localization, as
demonstrated here for ClC-4 and ClC-5, serves a physiological role or
is the by-product of a recycling via the plasma membrane. In any case,
the surface expression allows a biophysical analysis of channels whose
primary function may be in intracellular acidification.
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ACKNOWLEDGEMENTS |
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We thank K. Steinmeyer and B. Schwappach for several ClC-5 mutants and for critical reading of the manuscript, P. Haussmann, S. Lokitek, and B. Merz for technical assistance, and J. Hume and B. Horowitz for the gift of the guinea pig ClC-3 clone.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 49-40-4717-4741;
Fax: 49-40-4717-4839; E-mail:
jentsch{at}plexus.uke.uni-hamburg.de.
The abbreviations used are: CLC, chloride channel of the CLC gene family; ClC-X, member X of the CLC gene family; HEK, human embryonal kidney; GFP, green fluorescent protein; MES, 2-(N-morpholino)ethanesulfonic acid; TAPS, 3-[tris(hydroxymethyl)methyl]aminopropanesulfonic acid; NMDG, N-methyl-D-glucamine; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; WT, wild-type.
2 N. Wills, personal communication.
3 S. Schaffer, T. Breiderhoff, and T. J. Jentsch, unpublished results.
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REFERENCES |
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