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INTRODUCTION |
The pore architecture of anion channels is still poorly
known. Structure-function studies have been undertaken for a number of
structurally unrelated chloride channel classes, such as cystic fibrosis transmembrane conductance regulator (1, 2),
ligand-gated anion channels (3), and CLC channels (4). Sedimentation studies suggested that ClC-0 (5), ClC-1 (6), and a bacterial CLC
protein (7) are dimers. For the bacterial CLC, this was confirmed by
cross-linking experiments. In single-channel recordings, ClC-0 displays
two conductance levels of equal magnitude. These gate independently,
but are shut off together by a different, slow gating process. This led
to the suggestion that ClC-0 is a "double-barreled" channel, which
has two identical, largely independent pores (8). This model was
confirmed by studies in which only of the subunits in the homodimer was
mutated (9, 10). These channels displayed single-channel conductances
that were compatible with one wild-type pore and one mutated pore. The
important question if each subunit individually forms a pore has also
been addressed for ClC-0. Concatemers with two mutant subunits
suggested that one subunit forms one pore (10), although the presence
of two mixed pores, formed by different parts of each subunit, could
not be completely ruled out. However, the double-barreled structure of
CLC channels has recently been questioned. The effect on whole
cell-currents caused by the modification of cysteines in mutant ClC-1
channels led to the suggestion (11) that the two subunits of ClC-1 form
a single pore that includes the D3-D4 region from each subunit. In
single-channel records, however, ClC-1 displays a double-pore behavior
comparable to that of ClC-0 (12). All CLC channels identified so far
are homologous in the entire segment encompassing the 10-12
transmembrane domains. Hence, a common pore architecture must be
assumed. This implies that either the "one-subunit/one-pore" model
postulated for ClC-0 is valid for all members of this gene family, or
it is valid for none of the channels, including ClC-0.
To demonstrate the functional and structural separation of individual
pores in a CLC channel dimer, we constructed concatemers of two CLC
channel monomers, linked in a head-to-tail fashion. The expression of
concatenated subunits rather than coexpression of the corresponding
monomers offers the advantage that only a single type of dimer will be
formed. This approach has been used in the past to demonstrate separate
pores in the ClC-0 channel (9, 10, 13). We now extend it to the study
of concatemers composed of different CLC monomers, namely those of
ClC-0, -1, and -2. Both ClC-1 and ClC-2 are highly homologous to ClC-0,
with 54% and 49% identity at the amino acid level, respectively (14, 15). ClC-1 and ClC-2, which share 55% of sequence identity (15), have
already been shown to form functional mixed dimers with altered properties in coexpression experiments (16).
Since ClC-0 has been studied extensively on the single-channel level
(8, 13, 17, 18), its presence in the mixed channels may be demonstrated
unambiguously by single-channel analysis. Only one study (12) showed
single-channel recordings of ClC-1, and single ClC-2 channels have not
yet been reported. In mixed concatemers of ClC-0, -1, and -2, we
observed properties of both constituent pores in the macroscopic
current. In single-channel recordings, we could clearly distinguish two
different conductance levels that can be attributed to the pores of the
constituent subunits. This demonstrates that one CLC subunit forms one
pore, which retains most of its properties in a dimer irrespective of its partner. This is also the first time that single-channel traces of
the ClC-2 pore are reported.
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EXPERIMENTAL PROCEDURES |
Construction of Concatemeric Channels and Expression in Xenopus
Oocytes--
To generate concatemeric channels, the stop codon of the
N-terminal subunit was replaced with a PacI restriction
site, which was then used to link it to the C-terminal subunit. The
linker sequence consisted of four amino acids (L-I-K-A). Point
mutations were introduced by recombinant
PCR1 and verified by
sequencing. Constructs were expressed in the pTLN vector (16), and
capped cRNA was transcribed in vitro with the mMessage
mMachine kit (Ambion, Austin, TX). 8-10 ng of cRNA were injected into
Xenopus oocytes as described (19), and measurements were
performed 2-4 days after injection, with mock-injected oocytes as controls.
Electrophysiology--
Voltage-clamp measurements were performed
using a conventional two-electrode voltage clamp (Turbo Tec 01C, npi,
Tamm, Germany) in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4). For
inhibitor measurements, oocytes were continuously superfused with ND96
containing varying concentrations of 9-AC, up to the solubility limit,
which was ~4 mM at neutral pH. Voltage protocols were
generated using pClamp software (Axon Instruments, Foster City, CA).
Patch clamp measurements were performed in the excised inside-out mode
after manual removal of the vitellin envelope. Patch pipettes of
2-3-megohm resistance were made from aluminosilicate glass
(Hilgenberg, Malsfeld, Germany), coated with Sylgard (General Electric,
Waterford, NY), and filled with 100 mM NMG-Cl, 5 mM MgCl2, 5 mM HEPES, pH 7.4. The
bath solution contained 106 mM NMG-Cl, 2 mM
MgCl2, 2.5 mM EGTA, 5 mM HEPES, pH
7.4, resulting in a symmetrical chloride concentration of 110 mM. Data were acquired with an Axopatch 200B amplifier
(Axon Instruments) using pClamp software, low pass-filtered at 1 or 4 kHz, and recorded on digital tape or on a hard disc, with acquisition
rates of 2 and 10 kHz, respectively. For display purposes,
single-channel data were digitally filtered at 330 Hz.
Data Analysis--
To determine the IC50 values for
9-AC (Fig. 1C), the following function was used.
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(Eq. 1)
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c denotes the inhibitor concentration and
Imin the current remaining at saturating
inhibitor concentration. For the mixed concatemer with two inhibitor
binding sites, the following function was fitted to the data.
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(Eq. 2)
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I1 is the current remaining at saturating
inhibitor concentration for the high affinity binding site and
Imin the current remaining at saturating
concentration for the low affinity binding site.
Single-channel current amplitudes were calculated from Gaussian fits to
all-points histograms. Mean open times were obtained from exponential
fits to dwell-time histograms. Fitting was done using the Origin
analysis software (Microcal Software, Northampton, MA).
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RESULTS |
Mixed Concatemers of ClC-1 and ClC-0--
To test whether
different CLC subunits may associate to form mixed pores with novel
characteristics, or whether one pore is formed exclusively by one
subunit, we constructed mixed concatemers of two CLC monomers. For
simplicity, we will describe our results in the framework of the
one-subunit/one-pore model and evaluate alternative models in the
discussion section.
Using a four-amino acid linker sequence (see "Experimental
Procedures"), ClC-0 and ClC-1 were linked in both possible
orientations, i.e. ClC-1~ClC-0 and ClC-0~ClC-1. For
comparison, homomeric concatemers of ClC-1 and of ClC-0 with the same
linker sequence were constructed. All four concatemers could be
expressed functionally in Xenopus oocytes (Fig.
1A). Apart from a reduced
expression efficiency, no conspicuous differences were found between
concatemeric and monomeric ClC-0, in accordance with previous studies
of concatenated ClC-0 (10, 13), which reported wild-type behavior for
macroscopic and single-channel properties of the concatemer. This
indicated that concatemerization per se did not alter
channel properties.

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Fig. 1.
Macroscopic properties of mixed concatemers
of ClC-0 and ClC-1. A, families of current traces,
obtained with the voltage protocol depicted in the insert, are shown
for the four possible combinations of concatemers consisting of ClC-0
and/or ClC-1. The ClC-1 concatemer clearly exhibits the properties of
ClC-1, yet the two mixed concatemers are indistinguishable from the
ClC-0 concatemer. B, the ratio of the tail current at +40
mV, obtained after a 7-s prepulse to the indicated potential, to the
current obtained with a +40-mV prepulse is a measure of the
hyperpolarization-activated slow common gate of ClC-0 (29). When this
is compared for the different concatemers, the ClC-0 concatemer shows a
considerable activation starting at ~ 60 mV, whereas both mixed
concatemers lack this activation by hyperpolarization. C,
the sensitivity to the inhibitor 9-AC is shown for three concatemers
consisting of ClC-1 and/or ClC-0(K519E) subunits, which have almost
equal single-channel conductances. The ClC-0(K519E) concatemer has an
apparent IC50 of 9.8 ± 0.1 mM, the ClC-1
concatemer of 8.2 ± 1.0 µM. In the mixed
concatemer, a small fraction of the current (about 12%) is inhibitable
by small concentrations of 9-AC (IC50 = 10.6 ± 11.7 µM) and the remainder is inhibited by much higher
concentrations (IC50 = 10.7 ± 3.4 mM).
Note that the 9-AC dependence of the 1~0 current, in contrast to
those of the homodimeric concatemers, is not well fitted by the
function used to determine the IC50. Data points in
B and C represent the mean ± S.E. of three
to six individual determinations. Error bars
smaller than the symbol size are not shown.
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The current amplitudes for the different concatemers differed
significantly at the peak of expression (3 days after RNA injection). The slope conductance at 0 mV of the 0~0 concatemer (116 ± 23 µS) was about 4-5 times higher than for the 1~1 concatemer
(26 ± 6 µS), and the two mixed concatemers had conductances of
67 ± 21 µS in the 1~0 and 159 ± 24 µS in the 0~1
orientation (n = 10-21). Protein levels were not
measured, so the differences in steady-state current amplitudes may be
caused either by a different conductance or by differences in
expression level. In general, however, current amplitudes increased
with the number of ClC-0 subunits in the concatemer, consistent with
the higher single-channel conductance of ClC-0 (8 pS (Ref. 19)) as
compared with ClC-1 (1.2 pS (Ref. 12)).
Both ClC-0 and ClC-1 are voltage-gated in more than one way. This has
been described as a "fast" gate and a "slow" gate for ClC-0 (8,
18). Although the slow gate in ClC-1 is much faster than in ClC-0, the
same terminology has been used for ClC-1 by Pusch and co-workers (12).
The voltage dependence of the fast gate is qualitatively similar for
ClC-0 and ClC-1. Both channels are closed by hyperpolarization, with
the midpoint of the activation curve at
100 mV for ClC-0 (20) and
20 mV for ClC-1 (21) in the oocyte system. Normal gating of ClC-1 is
retained in the ClC-1~ClC-1 concatemer. However, no ClC-1-like
currents were seen in the mixed concatemers with ClC-0. When a fast
voltage protocol was used, their currents were very similar to ClC-0
(Fig. 1A). The steady-state current voltage dependence of
the 1~0 and 0~1 concatemers was indistinguishable from that of the
0~0 concatemer (data not shown). Because of its lower single-channel
conductance, the contribution of ClC-1 to the macroscopic current is
expected to be small in mixed concatemers with ClC-0 (~10%), but it
should still be detectable. Although indistinguishable in their fast gating, both mixed concatemers differ from the ClC-0 concatemer by the absence of the hyperpolarization-activated slow gate, indicating that a different channel than in the 0~0 concatemer is formed (Fig.
1B).
The conclusion that the ClC-1 pore does not contribute significantly to
macroscopic currents of the mixed concatemers is further supported by
currents obtained from concatemers bearing the K519E mutation in the
ClC-0 pore. Although the single-channel conductances of ClC-1 (1.2 pS
(Ref. 12)) and ClC-0(K519E) (1 pS (Ref. 10)) are about equal, the
macroscopic current of the mixed concatemers was very similar to the
current obtained with the ClC-0(K519E) homodimer in terms of
steady-state voltage dependence and open channel rectification. This
was true irrespective of the order of the two subunits in the mixed
concatemer (data not shown). However, the differential sensitivity of
ClC-1 and ClC-0 to the inhibitor 9-AC (14, 22) may be exploited to
demonstrate the presence of a ClC-1 conductance in the 1~0(K519E)
concatemer. Extracellular 9-AC inhibited the ClC-1 concatemer with an
apparent IC50 of 8.2 ± 1.0 µM (Fig.
1C). This inhibition was not complete, because about 30% of
the current remained at 500 µM 9-AC. The ClC-0(K519E)
concatemer could only be inhibited by much higher 9-AC concentration,
with an apparent IC50 of 9.8 ± 0.8 mM. In the mixed concatemer, a biphasic inhibition was observed. A small fraction of the current (about 12%) was inhibited by similar
concentrations of 9-AC as was the ClC-1 (IC50 of 10.6 ± 11.7 µM), whereas the remaining current required
equally high 9-AC concentrations as the ClC-0(K519E) to become blocked
(IC50 of 10.7 ± 3.4 mM). This indicates
that the macroscopic current of the 1~0(K519E) concatemer is the sum
of two current components with the same 9-AC sensitivity as the ClC-1
and ClC-0(K519E), respectively. Contrary to expectation, the highly
9-AC-sensitive component is much smaller in amplitude than the
9-AC-insensitive component. This indicates that the activity of ClC-1
is reduced in the concatemer.
Since the properties of single ClC-0 and ClC-1 pores are known, it
should be evident from single-channel currents of the mixed concatemer
if both pores are present. Excised patches of 1~0 concatemers clearly
showed two different pores, a large pore with the typical properties of
a single ClC-0 pore and a small pore with properties of a ClC-1 pore
(Fig. 2A). These two pores
were invariably found together in the patch (12 out of 12 patches). The
single-pore current voltage relationship was linear in the range of
60 and
160 mV (
80 and
160 mV for the small pore). Mean
conductance of the small pore was 1.8 pS, about 50% larger than
reported for wild-type ClC-1 pore at a lower pH of 6.5 (12). The
conductance of the large pore was 7.8 pS and thus of the same magnitude
as in the ClC-0 homodimer (~8 pS) (Ref. 13 and data not shown).

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Fig. 2.
Single-channel properties of the
ClC-1~ClC-0 mixed concatemer. A, a continuous current
trace of a single channel of the 1~0 concatemer, recorded at 100
mV, is shown. Four current levels (arrows) resulting from
the presence of two different pores can be distinguished. The gating of
both pores is independent of each other, because all possible gating
transitions are observed between the four levels with equal likelihood,
regardless of whether the other pore is open or closed (see
"Results"). B, the single-pore conductance is
determined from the current-voltage relationship in the range 80 to
160 mV. The conductance of the small pore (triangles) is
1.8 ± 0.1 pS; that of the large pore (squares) is
7.8 ± 0.2 pS, calculated from a linear fit to the data. Data
points represent the mean ± S.E. of 3-12 individual
determinations.
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Dwell-time analysis was performed in three patches with a stable
single-channel at
100 mV. The large pore had a mean open time of
34 ± 2 ms, which is in the range reported for individual pores in
the ClC-0 dimer (17, 18). The small pore had a mean open time of
29 ± 2 ms, slightly shorter than that of individual pores in the
ClC-1 dimer at pH 6.5 (~45 ms at
140 mV (Ref. 12)). This difference
is expected because a lower pH slows the overall gating of ClC-1 (23).
Very long closed times that lead to the bursting behavior normally
associated with ClC-0 single-channel currents were not observed in the
mixed concatemer.
Even in the absence of a clearly visible common gate, the gating of the
pores might be interdependent. We therefore determined the open
probability of the large pore in the 1~0 concatemer in relation to
the open state of the small pore. The single-channel record was
subdivided into sections of small pore open and closed events, and the
open probability of the large pore calculated under both conditions.
When the small pore was closed, the open probability (at
100 mV) of
the large pore was 0.45 ± 0.03 (n = 5 patches).
When the small pore was open, a value of 0.48 ± 0.02 was
obtained, suggesting that the gating of the large pore did not depend
on the open state of the small pore. The large pore open probability
agrees with the corresponding value for individual pores in homomeric
ClC-0, which is ~0.45 at this voltage (10).
Mixed Concatemers of ClC-2 and ClC-0--
The gating of ClC-2
differs significantly from ClC-1 and ClC-0. It opens very slowly upon
hyperpolarization, is virtually closed at positive potentials, and can
be opened by cell swelling and extracellular acidification (24, 25). An
N-terminal inactivation domain (residues 21-39) was proposed to
influence channel gating from the cytoplasmic side by a ball-and-chain
mechanism (24). It is currently unclear how many of these inactivation
domains are needed to gate a channel dimer. Possible movement
restrictions of the second inactivation domain in the 2~2 concatemer
apparently did not interfere with normal gating (compare Fig.
3A). However, when we
generated mixed concatemers of ClC-2 and ClC-0, only the concatemer
with the N-terminal ClC-2 moiety could be expressed functionally. In
stark contrast to the ClC-1/ClC-0 concatemers, where the ClC-0 pore
dominated the macroscopic current, current traces obtained with the
ClC-2~ClC-0 mixed concatemer showed only a small
depolarization-activated ClC-0 type conductance and a rather large
hyperpolarization-activated ClC-2 type conductance (Fig.
3A).

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Fig. 3.
Macroscopic properties of mixed concatemers
of ClC-0 and ClC-2. A, families of current traces,
obtained with the voltage protocol depicted in the insert, are shown
for the 2~2 and 2~0 concatemers. B, steady-state
current-voltage relationships for the traces shown in A. The
2~2 concatemer yields only hyperpolarization-activated currents, the
2~0 concatemer can be activated both by depolarization and by
hyperpolarization. C, in the 2~0 concatemer, the
hyperpolarization-activated current is sensitive to external pH. The
current shown is the tail current at +40 mV after a prepulse to the
indicated potential, corrected by the current obtained with a +40-mV
prepulse and normalized to the current amplitude under control
conditions ( 100 mV prepulse, pH 7.5). Data points represent the
mean ± S.E. of 5-12 different determinations.
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To investigate whether the hyperpolarization-activated current was
indeed carried by ClC-2 pores, we tested its modulation by external pH.
The current increased by ~40% upon lowering the pH by 1 unit, and
decreased by the same amount upon raising the pH by 1 unit (Fig.
3C). This is comparable to the pH sensitivity of ClC-2
wild-type currents, which are increased/decreased by ~60% upon
lowering/raising the pH by 1 unit (25). ClC-0 is also weakly dependent
on extracellular pH, but the moderate changes of ±1 pH unit employed
here would not affect the steady-state current at the test potential of
+40 mV (Ref. 17 and results not shown). This demonstrates that
pH-dependent activation, which is a characteristic feature
of ClC-2, is preserved in the 2~0 mixed concatemer.
Mutational analysis was used to identify the contribution of either
pore to the macroscopic current. To this end, point mutations in either
of the two subunits were inserted into the 2~0 concatemer and the
resulting changes in the macroscopic current analyzed (Fig.
4). The ClC-2(K210Q) mutation accelerated
the gating of homomeric ClC-2, resulting in a faster inactivation at
depolarized potentials (data not shown). Inserting this mutation in the
2~0 concatemer (Fig. 4, left panel) accelerated
the decay of the hyperpolarization-activated current upon switching to
positive voltages. The K210Q mutation also reduced the
hyperpolarization-activated current relative to the current at neutral
potentials. This was determined from the ratio of the slope
conductances at
120 mV and
40 mV, which was 2.43 and 1.33 for the
traces shown in Figs. 3 and 4, respectively. A different effect was
seen with the ClC-2(K566E) mutation, which caused outward rectification
of the open-pore currents in the homomer (25). This effect is preserved
in the mixed concatemer (Fig. 4, middle panel),
since the current at negative voltages is significantly reduced in
comparison with wild-type 2~0 currents. Finally, the ClC-0-like
current could be suppressed by the introduction of the ClC-0(K519E)
mutation, which greatly reduces the single-channel amplitude (10). The
current of the 2~0(K519E) concatemer is only slightly different from
that of the 2~2 concatemer (Fig. 4, right
panel), indicating that a ClC-2 pore with wild-type gating behavior is present in the mixed concatemer.

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Fig. 4.
Macroscopic properties of mutant
ClC-2~ClC-0 concatemers. A, families of current
traces obtained with the protocol shown in Fig. 3A for three
different point mutations in the 2~0 concatemer. B,
steady-state current-voltage relationships for the traces shown in
A. The ClC-2(K210Q) mutation accelerates the decay of the
hyperpolarization-activated current. The ClC-2(K566E) mutation renders
the current outwardly rectifying. Finally, the ClC-0(K519E) mutation
greatly reduces the depolarization-activated current, resulting in a
conductance resembling that of the ClC-2 homodimer (compare Fig.
3).
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Single-channel analysis of the 2~0 mixed concatemer is complicated by
the fact that no single-channel currents of ClC-2 have been published.
Macroscopic currents suggest that the ClC-2 pore should be open only at
negative potentials, and noise analysis indicated a single-channel
conductance of 2-3 pS (16). When single-channels of the 2~0
concatemer were recorded (Fig. 5), a pore
conforming to these predictions was indeed found in association with a
ClC-0 type pore. In all recordings that showed a single ~8-pS
conductance level, a smaller conductance of 2.8 pS was also found (7 patches). In contrast to the ClC-0 pore, which was always active and
gated rapidly, the small pore opened only slowly after switching from
positive to negative potential and closed quickly upon returning to
positive potential. Once opened by negative voltage, very long open
times, interrupted only by brief closings, could be observed (Fig.
5A, bottom trace).

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Fig. 5.
Single-channel properties of the
ClC-2~ClC-0 mixed concatemer. A, current traces of a
single channel of the 2~0 concatemer, recorded at 100 mV and 120
mV, are shown. The zero current level is indicated by dashed
lines. In addition to the fast-gating ClC-0 pore, a slow-gating smaller
pore is also present. At 120 mV, this pore is almost constantly open
and shows frequent but very short closings. B, the
single-pore conductance is determined from the current-voltage
relationship in the 80 to 160 mV range. The conductance of
the small pore (triangles) is 2.8 ± 0.0 pS, and that
of the large pore (squares) is 8.4 ± 0.2 pS,
calculated from a linear fit to the data. Data points represent the
mean ± S.E. of two to five individual determinations.
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To finally ascertain the identity of the small pore in the 2~0
concatemer, single-channel recordings of the 2~2 concatemer were
performed. This revealed pores with a single-channel conductance of
2.6 ± 0.1 pS (Fig. 6) that gated
similarly to the small pore in the 2~0 mixed concatemer. Again,
positive voltage caused the pores to close, and upon switching to
negative voltage, the pores re-opened only after a significant delay
(compare Fig. 6B). Unlike the ClC-0, where the slow gating
mechanism closes both pores simultaneously, ClC-2 showed no bursting
behavior. This constant channel activity of ClC-2, in combination with
its slow activation after a hyperpolarizing voltage step, did not allow
us to unequivocally determine the minimum number of active pores,
i.e. the pore stoichiometry of the homomeric channel.

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Fig. 6.
Single-channel properties of the
ClC-2~ClC-2 concatemer. A, a current trace of a patch
most likely containing two channels (four pores), recorded at 120 mV,
is shown. The five equidistant current levels are indicated by
dashed lines. Note the relatively slow gating of
the individual pores. B, current trace of the same patch,
after a switch from 0 mV to 160 mV. Only one pore opens initially,
interrupted by brief, flickery closings, until, after a delay of ~10
s, additional pores are activated. The dashed
line indicates zero current.
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 |
DISCUSSION |
Ever since the first double opening of the Torpedo
electric organ voltage-gated chloride channel appeared on the chart
recorder, the question whether this functional duplicity corresponded
also to a structural duplicity, i.e. a two-pore channel, has
been under debate. For the Torpedo channel, ClC-0, it has
been answered in favor of two separate conduction pathways in terms of
gating behavior (8, 17) and inhibitor blockade (26). The most stringent proof for this "double-barreled" model comes from the analysis of
point mutations in mixed concatemers (9, 10, 13). On the other hand,
the existence of a common gate that affects both pores simultaneously
in ClC-0 (8, 27) and ClC-1 (12), together with dominant negative
mutations in ClC-1 that alter channel gating (21) and dominant negative
effects of biochemical modification of single monomers in mixed ClC-1
concatemers (11), suggest a functional interaction between both halves
of the dimer. The cysteine modification studies of Fahlke and
co-workers (11) were even taken as direct evidence for of a single
conduction pathway in the dimeric channel, although neither true pore
properties nor single-channel behavior were investigated.
The question we have asked is this: are pore properties such as
single-channel conductance retained when subunits of different channels
are expressed together in a single dimer? If they are, the double-pore
arrangement observed in ClC-0 will be extended to other CLC channels.
In addition, this will show that the permeation pathway is completely
contained in a single subunit of the CLC dimer, as opposed to being
lined by parts of either subunit. In mixed concatemers containing
ClC-0, we have found that the smallest channel unit consisted of a
single ClC-0 pore accompanied by a smaller pore. The small pore behaved
like a ClC-1 pore in the 1~0 concatemer and like a ClC-2 pore in the
2~0 concatemer. Neither pore was encountered alone, but both pores
were invariably found together. Moreover, the gating kinetics of the
individual pores closely mimic the gating observed in homodimers of the
respective subunit. This means that the structures responsible for pore
formation and for voltage-dependent (fast) gating are
present in any one subunit.
Other properties, however, are dependent on both pores in the dimer.
This is clearly the case for the slow gate of the ClC-0, which is no
longer seen in the macroscopic current if one ClC-0 subunit is replaced
with a ClC-1 subunit. In the single-channel records of the mixed
concatemers, two types of coordinated gating activity may be discerned,
the direct transition between fully open and fully closed states, and
the direct transition between small and large open levels. The former
is nothing else but the slow gating well known in ClC-0, but the latter
interlevel transitions would be missed in homomeric channels with two
pores of equal conductance. Close inspection of the traces shown (Figs.
2A and 5A) seems to yield a few examples of
either type of gating, i.e. coordinated opening/closing
events as well as interlevel transitions. Considering the limited
bandwidth of our recordings (2 kHz prior to filtering), these could
result from incompletely resolved sequential gating events, but we
cannot rule out the possibility that coordinated gating activity takes
place in the mixed dimers with a low incidence. Since the frequency of
these events is in any case too low to significantly alter channel
behavior, we have not systematically investigated this example of
subunit interdependence.
Furthermore, the contribution of the ClC-1 pore is very much reduced
under voltage-clamp conditions when expressed alongside the ClC-0 pore,
although both pores are clearly active in excised patches. This
discrepancy may be due to the interaction with cytoplasmic cofactors or
depend on the low [Cl
] of the oocyte interior.
Interestingly, in earlier studies of ClC-1 and ClC-2 coexpression (16),
ClC-1 contributed little to the macroscopic current (and this most
likely resulted from ClC-1 homodimers). Rather, the macroscopic current
resembled that of a constitutively open ClC-2 channel. Quite different
from the apparent suppression of ClC-1 in the 1~0 and 0~1
concatemers, ClC-2 dominates the macroscopic current in the 2~0
concatemer under whole cell conditions, indicating that ClC-0 is
suppressed. Again, in the excised patch, ClC-0 and ClC-2 pores show
normal gating.
Assuming that two separate pores are present in a channel consisting of
two subunits, the question remains whether one pore is contained
completely within a single subunit or formed by parts of each subunit.
This question has been addressed in the past in concatemers of ClC-0
carrying two different mutations (10). Although these experiments were
fully compatible with a one-subunit/one-pore arrangement, they could
not entirely resolve the issue, because the pore structure of CLC
channel is not known. The experiments with dimers of two different CLC
channels that are reported here demonstrate that the basic channel
properties of the monomer are not altered by its interaction with other
subunits. This shows that a pore is formed entirely by a single CLC monomer.
We have interpreted our results based on the assumption that only two
CLC subunits are required to form a functional channel, but is this
justified? Our assumption is well supported by biochemical evidence,
which suggests a dimeric structure for ClC-0 (5), ClC-1 (6), and a
bacterial CLC homologue (7, 28). Nevertheless, a dimerization of the
concatemers used in this study (resulting in a dimer of dimers) cannot
strictly be ruled out. If this should happen, dimers of the two
constituent pores rather than mixed dimers could be formed, rendering
the single-channel studies meaningless. The presence of single pores of
each constituent channel type in the mixed dimers, however, argues
against this possibility, for in a tetrameric arrangement, four pores
should be present.
Can our results be explained in terms of a single-barreled channel, in
which both subunits contribute to a single pore? This alternative model
of CLC pore architecture was brought forward by Fahlke et
al. (11) based on the interaction of single cysteine mutants of
ClC-1 with mono- and bifunctional reagents. In the framework of this
model, the two equal-sized conductance values observed in
single-channel recordings must be regarded as subconductance states of
a common pore. A pore consisting of two different CLC proteins, as is
the case in our mixed concatemers, could then have two different
subconductance states. However, it seems impossible that such
subconductance states retain their conductance levels and gating
properties they have in the respective homodimer, and that they gate
independently of each other in the asymmetric heterodimer.
Taken together, our results argue for a common structural basis of all
CLC channels, with a separate conduction pathway, i.e. a
pore, in each subunit. The fundamental characteristics of channel activity, namely permeation of ions, mirrored in a defined
single-channel conductance, and voltage-dependent gating
transitions, are present in the monomeric channel and do not depend on
the partner subunit. Any CLC dimer, therefore, must be viewed as an
association of two basically independent pores. This does not exclude
the possibility that some CLC channels are monomers. However, since the
dimeric structure found in the bacterial channel (7) appears to be conserved in the mammalian channels, this seems unlikely. Last but not
least, we have shown on the single-channel level that ClC-2 is a slowly
gating, hyperpolarization-activated channel of 2-3 pS single-channel
conductance, in agreement with prior studies of macroscopic currents.
This enables a comparison with single-channel recordings from native
tissues in which ClC-2 is expressed.
What are the consequences of the one-subunit/one-pore arrangement? One
important feature of such a pore architecture is the mechanism by which
mutations may affect channel function. In potassium channels, where
four subunits contribute equally to a single pore, mutations in the
pore as well as in other parts of the protein often have dominant
negative effects. On the other hand, mutations in CLC channels will
only show a dominant phenotype if they affect a common gating mechanism
or if they lead to a retention or misprocessing of heteromeric channels
before they reach their target membrane. This observation is consistent
with the analysis of a dominant negative mutation in ClC-1 causing
myotonia congenita (12), which was found to affect the common (slow)
gating but not the individual (fast) gating of the channels.
Elucidating channel structure may therefore be an important tool for
the understanding of mechanisms of pathogenesis in human inherited
diseases. The double pore arrangement further implies that the design
of dominant-negative mutants, which could be useful in cell biological
or transgenic approaches, will not be an easy task for all members of
the CLC family.