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INTRODUCTION |
The main chloride channel of human skeletal muscle,
hClC-1,1 is a member of the
ClC chloride channel family that is unrelated to any other known ion
channels (1). The first membrane topology model of ClC proteins was
derived from hydropathy analysis (2); several times it had to be
revised on the grounds of firmer experimental evidence (3-5). Certain
mutations in the human ClC-1 gene (reviewed in Ref. 6) lead to
myotonia, a disease characterized by muscle stiffness. The study of
such myotonia-causing mutations provided first insights into the
relationships between the primary sequence and the functions of this
channel (7-10). Strong evidence suggests regions between transmembrane
segments D3 and the end of D5 to participate in the pore-forming
structure (11). However, these results are not in agreement with the
most recently postulated topology model of ClC-1 (5). Furthermore,
studies of ClC-0 (reviewed in Ref. 1), a homologous chloride channel in
the electric organ of Torpedo, led to the suggestion of
additional protein parts being involved in forming the ion conducting pathway.
ClC channels most likely consist of two subunits (12), and in the case
of ClC-0, each subunit was proposed to contain a single pore (4, 13,
14). Recent evidence speaks against this "double-barreled" channel
model, at least in the case of ClC-1 (15).
We have found earlier that the exposure of hClC-1, stably expressed in
HEK-293 cells, to 1 mM Zn2+ leads to a massive
reduction of the conducted chloride current (16). The results were
compatible with the presence of at least two extracellularly accessible
zinc-binding sites and a direct effect on ion permeation,
e.g. by obstruction of the pore. The histidyl-reactive
diethyl pyrocarbonate (DEPC), applied from the outside, also reduced
the currents through hClC-1. The aim of this study was to identify the
target residues for these blockers and, thereby, to draw inferences
regarding the membrane topology and the potential location of
pore-forming structures. It is known from other proteins, that
Zn2+ binding sites are often made up of several and
possibly different amino acid side chains (17). Because these residues
(histidine, cysteine, or acidic amino acids) can be distant on the
primary sequence but close in space, we also hoped that their
identification would provide us with the first three-dimensional
information on the ClC-1 protein. Therefore, we individually replaced
candidate cysteine and histidine residues with alanine, expressed the
mutants in tsA201 cells, and characterized their sensitivity toward the blocking agents.
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EXPERIMENTAL PROCEDURES |
Construction and Expression of Mutants--
Site-directed
mutagenesis was performed using a polymerase chain reaction-based kit
(QuikChange, Stratagene). Mutant constructs contained in the expression
vector pRc/CMV were used for transfection of tsA201 cells as described
(18). Transient transfection was achieved using the calcium phosphate
precipitation method (19) with ~0.01 µg of plasmid DNA per
3-5 × 103 cells/cm2. To detect cells
expressing recombinant mutant clones after transfection, the tsA201
cells were co-transfected with a plasmid encoding the CD8 antigen and
incubated with polystyrene microbeads coated with anti-CD8 antibody
(Dynabeads M-450 CD8, Dynal GmbH, Hamburg, Germany) 3 min before
electrophysiological characterization.
Electrophysiology--
Two to three days after transfection,
standard whole-cell recordings were performed, using an EPC-7 amplifier
(List, Darmstadt, Germany). Only cells to which microbeads had bound
were used. For measurements of WT currents, a stably transfected
HEK-293 cell line was used (16) or tsA201 cells were transiently
transfected as described for the mutants. No differences were observed
with these two types of cells regarding electrophysiological
characteristics or blocker sensitivity. The standard external solution
contained 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4. For variation of pH, HEPES was
substituted on an equimolar basis with MES for pH 5.5-6.7 and with
AMPSO for pH 8.0-8.5. The recording pipettes were pulled from
borosilicate glass and were heat polished. They had resistances between
1.0 and 2.0 M
when filled with standard internal
solution (130 mM CsCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, pH 7.4). More than 60%
of the series resistance was compensated for by an analog procedure.
Linear leakage or capacitive currents were not subtracted. The currents
were filtered using the 3-kHz filter of the amplifier and sampled at
various rates. For recording and analysis of experimental data, a
combination of three programs (pCLAMP, Axon Instruments, Foster City,
CA; Excel, Microsoft, Unterschleissheim, Germany; SigmaPlot, Jandel Scientific, San Raphael, CA) was used. Differences of current reduction
were tested for statistical significance (p < 0.05) using one-way analysis of variance followed by the Tukey-Kramer multiple comparisons test (GraphPad InStat, San Diego, CA). All data
are shown as mean ± S.E.
The pH-dependence as well as concentration dependences of
Zn2+ block were fitted with logistic functions yielding
estimates of IC50, pKa, and Hill coefficients.
To determine the voltage dependence of activation, instantaneous tail
current amplitudes at
105 mV were normalized to the extrapolated
maximum value and plotted versus the test pulse potential, yielding the voltage dependence of the relative open probability, Popen (16). The data were fitted with a single
Boltzmann term plus a voltage-independent value:
I(V) = A·(1 +
exp[(V
V0.5)/Kv])
1 + constant.
Application of Reagents--
ZnCl2 (Sigma Chemical
Co., Deisenhofen, Germany) was prepared as a 100 mM stock
solution in standard external solution and diluted prior to the
experiments. All visible precipitates were eliminated by adding
sufficient HCl; no pH change was noted after dilution. DEPC (Fluka,
Buchs, Switzerland) was stored desiccated at 4 °C to minimize
decomposition by hydrolysis. A 200-mM stock solution in absolute
ethanol was freshly prepared every day, kept on ice, and diluted with
standard external solution immediately before use. Stock solutions were
added to the bath in the appropriate amounts, or else the cells were
superfused with the indicated reagent solution. To study the pH
dependence, the cells were exposed to bathing solutions of the desired
pH, followed by application of the blocking agent.
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RESULTS |
Dependence of Chloride Current Block by Zn2+ or DEPC on
pH--
First we tested the effect of external pH on WT current block
by external application of Zn2+ and DEPC. From the
pKa and Hill coefficient of such titration curves,
we hoped to infer the most likely sort and number of residues
participating in the block. Changes of pH alone have profound effects
on the gating of hClC-1, whereas outward current amplitudes do not
change considerably (20, 21). Block of whole-cell currents was tested
by applying repetitive pulses to +50 mV (for maximum channel
activation), followed by a hyperpolarizing step at
125 mV to monitor
the deactivation time course (Fig.
1A). Block by 1 mM
Zn2+ (registered at +50 mV) showed a strong pH dependence
(Fig. 1B). At pH 6.0, the current amplitudes were
unaffected, whereas at pH 7.4, the block was almost complete. The
apparent pKa of the resultant titration curve was
6.9, and the Hill coefficient was 3.0. In contrast, the effect of 1 mM DEPC was practically independent of pH over the range
tested, pH 5.5-8.5.

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Fig. 1.
Dependence of Zn2+
and DEPC block on the pH of the external bath solution.
A, examples of chloride current traces recorded in the
whole-cell mode from tsA cells expressing WT hClC-1 channels. The
control pulse program changed the membrane potential from its holding
value of 0 mV first to +50 mV for 100 ms and then to 125 mV for 400 ms and was repeated every 15 s. Solid line represents
trace obtained in standard bathing solution and dotted line
after external application of 1 mM Zn2+,
i.e. at the beginning and end point of blockade.
B, current block is expressed as proportion of the
amplitudes at +50 mV before (Imax) and after
(I) application of blocking agents and plotted against the
varied pH of the bathing solution (n = 1-6). Shown are
fractions obtained in the presence of 1 mM Zn2+
( ) or in the presence of 1 mM DEPC ( ).
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Functional Characterization of Mutants with Cysteine and Histidine
Replaced by Alanine--
According to the topology model of
Schmidt-Rose and Jentsch (Ref. 5; see Fig.
2A), the cysteine residues at
positions 242, 254, 271, 481, and 546, and the histidine residues at
positions 237, 436, 451, 538, and 555 are potentially accessible from
the extracellular environment. To test whether binding of
Zn2+ to either of these residues is involved in the
observed block, we individually replaced each of these residues by
alanine. Positions Cys-179 and His-180 were included in this test
because an alternative model placed D2 instead of D4 at the outside
(Ref. 4; not shown in figure).

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Fig. 2.
Membrane topology models of hClC-1.
A, model based on results from Schmidt-Rose and Jentsch (5).
The positions of all cysteine and histidine residues are indicated by
corresponding letters, and those of residues mutated in this
paper additionally are indicated by squares and
numbers. Segments corresponding to the initially proposed
hydrophobic domains D1-D13 (2) are indicated by numbers.
B, model for D3-D5 region according to Fahlke et
al. (11).
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All mutants were functional when expressed in tsA201 cells, and the
conducted whole-cell currents were not substantially different from WT
currents. They were of similar amplitude and showed the typical
deactivation in response to hyperpolarizing pulses as well as inward
rectification (Fig. 3).

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Fig. 3.
Whole cell current recordings from WT as well
as from cysteine and histidine mutants. A, current
traces from WT (middle) elicited by the pulse protocol on
the left and deduced voltage dependence (right)
of normalized instantaneous (circles) and late current
(squares) amplitudes, i.e. at the beginning and
end of the test pulse (n = 7). In the middle
panel, every second trace has been omitted for clarity. Current
traces of all cysteine (B) and histidine (C)
mutants analogous to those shown in panel A for WT. Data for
constructs C242A/C254A/H180A/H451A (=CCHH/A) are
included in panel C; CCC/A = C242A/C254A/C546A. Voltage dependence of instantaneous and late current
amplitudes closely resembled that of WT as seen in Fig. 3A,
right (n = 4-11, data not shown).
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Sensitivity of the Cysteine and Histidine Mutants to
Zn2+--
In the case of the cysteine mutants C179A,
C271A, and C481A, the amount of chloride current block exerted by 1 mM Zn2+ was very similar to that in WT (Fig.
4A). In contrast, block was
almost absent with C546A (current reduction only by 17% instead of
83% in WT). Moderate changes (current reduction by 66-67%) were
observed in the case of mutants C242A and C254A. These residues are
located in segment D4 (see Fig. 2). The location of this segment is yet
unclear, as evidence based on substituted cysteine accessibility suggests it to face (at least in part) the cytoplasm (11), whereas topology studies predict it to be extracellular (5). Furthermore, residues following the C-terminal end of D3 (designated P1) and in D5
have been identified to participate in the pore-forming structure
(11).

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Fig. 4.
Block of chloride current effected by
Zn2+ and DEPC. For WT and each mutant, the
maximum reduction of current is expressed on the left
axis in percent of the initial current amplitude at +50 mV
( 100%, I = Imax, see Fig.
1A; n = 3-8). On the right axis,
degree of block is plotted (100% no current left). Block by 1 mM Zn2+ for (A) cysteine and
(B) histidine mutants. Data for construct
C242A/C254A/H180A/H451A (=CCHH/A) are included in
panel A. Block of histidine mutants by 1 mM DEPC
is shown in panel C. *, differences statistically
significant compared with WT.
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To test further whether Zn2+ binds to Cys-242 and Cys-254,
we constructed a double mutant (C242A/C254A) and investigated its susceptibility to Zn2+ blockade. In standard solutions the
electrophysiological characteristics of the double mutant resembled
that of WT (Fig. 3B). In the presence of 1 mM
Zn2+, the chloride currents were only decreased by 33%
(instead of 83% in WT), confirming the putative capabilities of these
two cysteines to bind extracellularly applied Zn2+ and
thereby establish channel blockade.
When mutants H436A, H538A, and H555A were in the same way externally
exposed to 1 mM Zn2+, reduction of current was
about the same as with WT (Fig. 4B). Currents through the
mutants H180A and H451A were slightly less blocked by Zn2+
(statistically significant) and, interestingly, in the case of H237A,
Zn2+ was able to block the chloride currents even by 6%
more than in WT. Also, the time course of block was markedly faster in
this case (data not shown).
To test for a possible role of His-180 and His-451 in the blocking
effect of Zn2+, we expressed the mutant H180A/H451A, and
additionally C242A/C254A/H180A/H451A, to possibly destroy the binding
site completely. The double histidine mutant showed a very similar
sensitivity toward Zn2+ as the two corresponding single
mutants (block by ~70%), whereas for the quadruple mutant, it was
about as much as with the double mutant C242A/C254A (block by ~33%).
This suggests that the two histidine residues are not involved in the
blockade exerted by Zn2+.
Concentration Dependence of Zn2+ Inhibition for Five
Cysteine Mutant Constructs--
To further investigate the changes in
Zn2+ block observed for C242A, C254A, and C546A, we
measured current block of these three mutants at different
Zn2+ concentrations. In addition, the double mutant
C242A/C254A and the triple mutant C242A/C254A/C546A were also studied.
The resulting curves are depicted in Fig.
5. The different degrees of
Zn2+ sensitivity already indicated by the values obtained
for 1 mM Zn2+ was fully confirmed. Whereas for
WT, an IC50 value close to 200 µM was
obtained, it took ~700-800 µM Zn2+ to
produce half-maximal block of the single cysteine mutants C242A and
C254A and ~1200 µM for the corresponding double mutant. The IC50 value for C546A was even larger (~1900
µM), and after removal of all three cysteines, a value
fifty times that of WT was obtained (~9200 µM). The
Hill coefficient was around 2.6 for WT, and it ranged from 1.6 to 2.5 for the single and double mutants. In the case of the triple mutant,
the value was reduced to 0.9.

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Fig. 5.
Concentration dependence of
Zn2+ block. The fractional current
amplitudes recorded at +50 mV at different Zn2+
concentrations are shown for WT, C242A, C254A, C546A, C242A/C254A, and
C242A/C254A/C546A. Lines represent fits with logistic
functions (n = 1-11).
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Sensitivity of the Histidine Mutants to DEPC--
We also tested
the responses of all histidine mutants to extracellularly applied 1 mM DEPC. This reagent preferably modifies histidine
residues by covalent binding of the carbethoxy group to the
unprotonated imidazole ring, although modification of other amino acids
has been described (22). The chloride currents through H180A, H436A,
and H451A were blocked by a similar degree as in cells containing WT
channels (47%, Fig. 4C). Block appeared stronger for
mutants H237A, H538A, and H555A (63%, 63%, and 74%, respectively), but the difference was significant only for the latter residue.
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DISCUSSION |
All 14 mutants that we have investigated were functional and
showed electrophysiological characteristics resembling those of WT
channels. Thus, we can presume that the structural integrity of the
channel protein was preserved in each case.
With mutants C242A, C254A, and C546A, the zinc block of the chloride
current was smaller than with WT. This suggests that the three
cysteines Cys-242, Cys-254, and Cys-546 are involved in the binding of
Zn2+, creating this effect. This result is surprising in so
far as the apparent pKa value of the
Zn2+ titration curve (pKa = 6.9, Fig.
1B) suggests that a histidine side chain
(pKa
6-7) rather than a cysteine (pKa
8-9 in proteins) is the relevant target
site. Studies on the pH dependence of ClC-1 current block by
Cd2+, another group IIb cation with behavior similar to
that of Zn2+, yielded a pKa value of
6.8, i.e. nearly identical to ours (23). The authors of that
study suggested that histidine residues might be involved. On the other
hand, considerable pKa shifts of titratable groups
are not uncommon in proteins, e.g. in our case because of
the presence of a positive charge close to the cysteine thiol.
None of the investigated histidine mutants showed a decreased
sensitivity toward the histidine-selective reagent DEPC. This could
either mean that DEPC has its target site located at a different position or that it exerts its effect by some unspecific mechanism. The
apparent lack of pH dependence of the DEPC effect does not allow one to
distinguish between these two possibilities.
Because the zinc blockade of WT channels can only be exerted by
external application of the metal ion (16), cysteines 242, 254, and 546 must be accessible from the extracellular environment. The finding that
the gating parameters were unchanged (16) also led us to assume that
Zn2+ might exert its blockade by a direct effect on
chloride permeation, e.g. by occlusion of the pore. Thus,
the three cysteine residues are presumably located in or near the pore
region of the channel. The degree of zinc block was markedly diminished
in the case of C546A, and only moderately in the case of the other two
mutants. Hence, Cys-546 might be closer to the pore than Cys-242 and
Cys-254. The latter two cysteines certainly influence the passage of
chloride through the pore when Zn2+ is bound because the
double mutant C242A/C254A is clearly less sensitive toward
Zn2+ as each of the single mutants alone.
Cysteines 242 and 254 are located in the D4 segment, and the residues
in and around this region have indeed been postulated to form the
channel-lining structure (11). However, when positions 237, 244, 265, and 267 were mutated to cysteines, they were only accessible to
internally applied methanethiosulfonate reagents (11). Therefore Fahlke
et al. (11) suggested D4 to be located at the inside of the
membrane (Fig. 2B). In contrast, Schmidt-Rose and Jentsch
(5), using glycosylation scanning and protease protection assays,
proposed D4 to be located on the extracellular side of the membrane. At
first glance, our results seem to speak in favor of the latter model.
In addition, the absence of a voltage dependence of the
Zn2+ blockade (16) and the fact that the ClC-1 pore is
forming an anion-selective channel lets the possibility of
Zn2+ penetrating this channel seem unlikely. However,
positively charged methanethiosulfonate reagents were able to reach
cysteine residues within the putative pore (11), and very recent
findings imply binding sites for internally applied Cd2+ at
some of these positions (15). Furthermore, work on the GABA receptor, a
chloride channel which is also Zn2+-sensitive, led to the
proposal that Zn2+ is gaining access to the anion channel
lumen, possibly in the form of the complex
(ZnCl)42
(24). The conformation and
orientation of the region between D3 and D5 might also be more complex,
e.g. looping through the membrane, and thereby exposing
parts of it to the outside and the inside. Thus, more experimental data
are needed to unambiguously assign a certain topology to this area of
the protein and to elucidate the pore-lining structure.
Another intriguing observation is that Zn2+ block of H237A
seemed to be stronger (and faster, data not shown) than in the case of
WT, although the difference was not statistically significant. The
histidine side chain at this position has been proposed to line the
channel lumen (11, 15). By substituting it with the smaller, uncharged
alanine side chain, the access for Zn2+ toward its actual
target sites (one or more of the three cysteines) nearby might be
facilitated. The same argument could explain the somewhat stronger
block of mutant H555A by DEPC (Fig. 4C).
The amount of block effected by Zn2+ in mutants H180A and
H451A is similar to that in mutants C242A and C254A. This led us
initially to think of a motif found in some zinc finger proteins: two
histidine and two cysteine residues surrounding a Zn2+ ion
in a tetrahedral arrangement (25). After removal of one of the binding
partners, Zn2+ might still be able to bind but with a lower
affinity. The lack of any additional blocking influence of the two
histidine mutants in the double mutant H180A/H451A and in the construct
C242A/C254A/H180A/H451A, however, argues against their potential
involvement in Zn2+ binding.
The mutation producing the largest difference in Zn2+
sensitivity was C546A. The proposed extracellular accessibility of
residue Cys-546 is in agreement with results suggesting that position Leu-549 is also accessible from the exterior (5). Furthermore, ClC-0
mutations situated before and at the end of D12 result in altered
permeation properties, implying a possible participation of these
regions in forming the channel-lining structure (5, 26).
It is unclear yet whether Cys-242, Cys-254, and Cys-546 participate in
the same binding site although the Hill coefficient (3.0) of the
titration curve seems suggestive. The substitution of all three
residues in the triple mutant C242A/C254A/C546A results in a channel
which is almost insensitive to Zn2+ blockade. The Hill
coefficient (~0.9) implies a loss of cooperativity and the removal of
one or more binding sites compared with WT (~2.6). In light of the
new evidence indicating that ClC-1 has a single pore (15), binding
sites made up of corresponding cysteines from both subunits are also plausible.