 |
INTRODUCTION |
The ClC family of voltage-gated Cl
channels
represents the largest known gene family coding for anion channels
(1-3). At least nine human isoforms (ClC-1 to ClC-7, ClC-Ka, and
ClC-Kb) are expressed in various tissues and play important roles in
the function of various organs. Mutations in the genes coding for three
ClC isoforms cause inherited human diseases. CLCN1
represents the genetic locus for myotonia congenita (4, 5), a muscle disease characterized by stiffness upon sudden forceful movement. Mutations in CLCN5 cause Dent's disease, an inherited renal
disorder associated with hypercalciuria, nephrolithiasis, and low
molecular weight proteinuria (6). Genetic alterations of
CLCNKB are responsible for type III Bartter's syndrome, a
salt-wasting renal tubular disorder causing hypovolemia,
hyponatremia, and hypotension (7).
Heterologous expression of many cloned ClC isoforms have revealed
highly anion-selective ion channels, and it is therefore reasonable to
predict that evolutionarily conserved structures confer anion
selectivity to their ion conduction pathways. Elucidating the molecular
mechanisms responsible for ion selectivity and conduction in ClC
channels is key to understanding the function and dysfunction of this
important family of ion channels. Moreover, primary sequence information about the ionic pore represents the first step for a
rationally designing compounds to block or open these ion channels that
play important roles in human diseases.
We recently probed the ion conduction pathway of human ClC-1 in the
presence of several different permeant anions (8), and we demonstrated
an unusual ion selectivity mechanism that depends upon differential ion
binding to discriminate among anions (8, 9). Human ClC-1
(hClC-1)1 exhibits at least
two functionally distinct ion binding sites within the pore (8, 10),
both preferring large and polyatomic anions over chloride. The tighter
binding of these anions reduces their turnover, resulting in lower
permeability and conductivity, thus making chloride the most permeant
anion (8, 9). This selectivity mechanism requires that binding sites
within the hClC-1 pore exhibit only a low affinity for permeating
anions to allow a measurable ion flow per unit of time. In agreement
with this prediction, an evaluation of the blocking action of iodide in hClC-1 revealed dissociation constants in the millimolar range (8, 10).
Because iodide binds more tightly than chloride, this value represents
a lower limit for the chloride dissociation constant and demonstrates
that ClC-type channels bind anions with affinities that are more than
an order of magnitude lower than the values with which potassium (11)
and calcium channels (12) interact with their respective permeant ions.
This result suggests that anions probably do not make intimate contact
with pore-lining residues during permeation through the hClC-1 pore.
The peculiar nature of anion selectivity in ClC channels poses a
particular problem for approaches using a combination of site-directed
mutagenesis and cellular electrophysiology to identify pore residues.
Allosteric and other long range effects of point mutations can have
considerable influence on the interaction between channel and permeant
anion and thus on selectivity and single channel amplitude. An effect
of a single amino exchange on ion conduction properties is therefore
not sufficient to establish that a region contributes to the formation
of the ion conduction pathway, and indeed point mutations at various
locations within the ClC channel sequence affect anion selectivity
(13-17). It is therefore necessary to use additional experimental
tests to identify pore-forming regions of ClC channels.
We have previously employed a combination of several methods to
identify two regions within hClC-1 that line a portion of the ion
conduction pathway and represent major structural determinants of ion
selectivity. A highly conserved 8-amino acid sequence present in every
known eukaryotic ClC channel (GKXGPXXH) lines the
most narrow part of the ClC pore. The P1 region is a major determinant of the binding affinities for anions (15) and is responsible for the
high anion to cation selectivity of hClC-1 (18). Within the fifth
transmembrane domain (D5), we have identified another highly conserved
amino acid region, GVLFSI in hClC-1 (designated as P2), that together
with P1 lines the narrow part of the of pore (18). In the present
study, we extend our investigation to another highly conserved segment,
the linker region between transmembrane domain 2 (D2) and 3 (D3). This
segment was previously demonstrated to have a single residue that can
influence single channel conductance of ClC-0 (16), but a systematic
evaluation of this region was lacking. We now provide further evidence
that this segment is important for ion selectivity, and we provide evidence that this region contributes to the formation of a wide inner
pore vestibule.
 |
MATERIALS AND METHODS |
Mutagenesis and Construction of Heterodimers--
Site-specific
mutants were constructed using recombinant polymerase chain
mutagenesis. Monomeric constructs were assembled in the expression
construct pRc/CMV-hClC-1, and dimers were constructed as described
previously (19). We then sequenced in both constructs regions modified
by polymerase chain reaction completely to exclude polymerase errors.
At least two independent recombinants were examined functionally for
each mutant or dimer. Transient transfection of tsA201 was performed as
described previously (15, 19).
Electrophysiology--
Standard whole-cell, inside-out, or
outside-out patch clamp recordings (20) were performed using an
Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Pipettes
were pulled from borosilicate glass and had resistances of 0.8-2.2
M
. More than 80% of the series resistance was
compensated by an analog procedure. The calculated voltage error due to
series resistance was always <5 mV. Currents were filtered with an
internal 4-pole Bessel filter with 1 kHz (
3 dB) and digitized with
sampling rates of 5 kHz using a Digidata AD/DA converter (Axon
Instruments, Foster City, CA). Cells were clamped to 0 mV for at least
5 s between two test sweeps. The compositions of the solutions
were as follows: extracellular solution (in mM), NaCl
(140), KCl (4), CaCl2 (2), MgCl2 (1), HEPES
(5), pH 7.4; intracellular (in mM), NaCl (130), MgCl2 (2), EGTA (5), HEPES (10), pH 7.4. Anion permeability ratios were determined as described previously (15). Data were analyzed
by a combination of pClamp (Axon Instruments, Foster City, CA) and
SigmaPlot (Jandel Scientific, San Rafael, CA) programs. All data are
shown as means ± S.E. from at least three different experiments.
Modification with MTS
Reagents--
2-Aminoethyl-methanethiosulfonate,
methylethiosulfonate-ethyltrimethylammonium (MTSET), and
-ethylsulfonate (MTSES) were obtained from Toronto Research Chemicals
(New York, Ontario, Canada). Stock solutions (0.1 M) were
prepared in distilled water, stored at
20 °C, and diluted into the
bath solution immediately before use. Cells/patches were held at 0 mV
and stimulated every 5 s to voltage steps to +75 mV followed by
105 mV. Typically, 20 cycles were employed to assess control values
(Icontrol). Then MTS-reagents were applied to
cells/patches by moving the cell/patch into the stream of a
silane-treated macropipette filled with MTS-containing solution. After
3 min or after reaching a steady-state current amplitude, cells/patches
were moved out of the stream to visualize reversible effects and to
measure the current amplitude after irreversible modification
(Imodified). The relative current reduction was
measured as 1
Imodified/Icontrol. The
time course of modification was fit with a single exponential giving
the time constant of modification. The pseudo-first order rate constant
was calculated as the inverse of the modification time constant.
Dividing by the concentration of the MTS reagents provided the second
order rate constants. The charge selectivity of the access pathway for MTS reagents was calculated by dividing the second order reaction rates
for MTSES by the rate for MTSET
(kMTSES/kMTSET) and
normalizing this value to the relative reactivity of the two reagents
with thiols in aqueous solution 0.08 (21). For experiments testing the
effect of SCN
on MTS modification, 50 mM NaCl
were substituted by equimolar amounts of NaSCN.
 |
RESULTS |
D2-D3 Linker Lines the Inner Mouth of the hClC-1 Pore--
All
known ClC channels exhibit a conserved sequence motif between
transmembrane domains D2 and D3 that has the sequence GSGIPEMK in
hClC-1 (Fig. 1). Earlier experiments
demonstrated that an amino acid exchange within this region (S123T)
changes the single channel amplitude and the conductivity sequence of
ClC-0 (16). The high degree of sequence conservation together with the
observed effect of a single point mutation on ClC-0 conduction
properties makes this segment (P3) a candidate pore-forming region. To
investigate further the role of P3 in formation of the ion conduction
pathway, we constructed single cysteine substitutions for each residue in the region and evaluated these mutants using the patch clamp technique and chemical modification with cysteine-specific
methanethiosulfonate (MTS) reagents.
Fig. 2 illustrates representative
macroscopic current recordings from each of the 8 single cysteine
mutants, obtained using either whole-cell (Fig. 2A) or
excised inside-out recordings from transiently transfected tsA201 cells
(Fig. 2, B-H). All mutants were functional in transiently
transfected tsA201 cells exhibiting large whole-cell current levels
with maximum outward current amplitudes of several nA. With the
exception of G188C, all mutants expressed at levels high enough to
permit recording of large amplitude macroscopic currents in excised
patches. Except for slight alterations in the voltage dependence of
activation and the time course of deactivation (data not shown), all
displayed gating properties very similar to wild-type (WT) hClC-1 (22).
In addition, all mutants exhibited an inwardly rectifying instantaneous
current-voltage relationship that is characteristic of hClC-1. We
conclude that cysteine substitutions within P3 do not disturb the
general functional behavior of hClC-1, and this provides evidence that
the mutations do not cause major structural rearrangements in the
channel.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2.
The effect of cysteine substitution within
the P3 region. Current recordings from mutant hClC-1 channels. For
each construct, voltage steps between 165 mV and +75 mV in 60-mV
steps were applied from a holding potential of 0 mV. Each test pulse is
followed by a fixed 105-mV pulse. Whole-cell recordings are shown for
G188C (A); and excised inside-out patches were used for
S189C (B), G190C (C), I191C (D), P192C
(E), E193C (F), M194C (G), and K195C
(H).
|
|
To test for alterations of the relative anion permeability sequence,
reversal potentials were determined from cells internally perfused with
standard internal solution and bathed in external solutions in which
140 mM NaCl was substituted with equimolar sodium salts of
Br
, I
,
NO3
, SCN
. Permeability
ratios were then calculated using the Goldman-Hodgkin-Katz equation
(15, 18) (Table I). Mutations of P3
residues affect the relative anion permeability ratios. These effects
are most dramatic for mutations G188C and S189C, whereas cysteine
substitutions at other locations only affect the relative thiocyanate
to chloride permeability ratio. Replacement of lysine 195 has only
minor effects on anion selectivity suggesting that this positively
charged side chain does not play a major role in hClC-1
selectivity.
Regions that contribute to the formation of an ion channel conduction
pathway contain residues that are in contact with the aqueous
environment. To test whether substituted cysteines within P3 project
their side chains into an aqueous cavity, we examined the mutants for
reactivity to hydrophilic cysteine-specific MTS reagents (18, 23). For
these experiments, patches/cells were held at 0 mV and stimulated every
5 s with a pulse consisting of a voltage step to +75 mV followed
by a
105-mV step. After 20 steps in standard solution, the patch/cell
was moved into a stream of a solution containing the MTS reagent at the
given concentration. The time course of the instantaneous as well as of
the late current amplitudes for the +75-mV and the
105-mV step were
determined by plotting the amplitudes versus the time since
the beginning of the MTS application. Three minutes later or after
reaching a steady-state current amplitude, patches/cells were moved out of the stream to visualize reversible effects. For WT hClC-1 channels, there is no change of any of these current amplitudes, neither by
internally nor externally applied MTS reagents (Fig.
3A) (18). Similarly, external
application of MTSES or MTSET does not have functional effects on any
of the eight cysteine-substituted channels, and this is compatible with
the predicted intracellular location of P3.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
Substituted cysteine accessibility
experiments. A, relative current change for substituted
cysteine between S189C and K195C following application of intracellular
MTSES (open bars) or MTSET (solid bars). For
G188C, G190C, and I191C the relative changes of the instantaneous
current amplitude at 105 mV are shown; for the remaining mutants the
relative current amplitude changes at the +75 mV step. B,
reaction rates for modification of these cysteines by MTSES or MTSET.
C, relative anion selectivity of the access pathway for MTS
reagents to four different substituted cysteines. All data were
obtained from excised inside-out (for application of MTS reagents to
the intracellular membrane side) or outside-out (for application of
MTS-reagents to the extracellular membrane side) patch clamp
recordings.
|
|
For internal application, MTS modification could be observed for S189C,
G190C, I191C, P192C, E193C, and K195C. Two types of functional
alterations could be observed in these experiments. For mutants G190C
and I191C, a time-dependent decrease of instantaneous as
well as of the late current amplitudes at +75 and
105 mV occurred after application of MTS components. The relative changes at the different voltages are similar, and this implies that there are no
MTS-induced changes of voltage-dependent gating, but rather the observed decrease of the current amplitude is caused by interaction of the reagents with the ion conduction pathway. For P192C, MTSES and
MTSET have opposite effects on the channel function. MTSES causes a
decrease of all measured current amplitudes, similarly to the results
obtained with G190C and I191C. In contrast, after application of MTSET
the instantaneous current amplitude at +75 mV increased, without
comparable changes of the late current amplitude at the same potential.
Whereas the instantaneous amplitude at
105 mV remained almost
unchanged, a change of the deactivation time course was observed at
this potential (Fig. 6A). These changes indicate a shift of
the activation curve of P192C to more negative potentials after channel
modification by MTSET. For mutant S189C, we also observed
time-dependent shifts of the voltage dependence of
activation that cause an increase of the current amplitude at certain
potentials. For E193C, there was a time-dependent decrease of the late current amplitude at +75 mV and the current amplitudes at
105 mV (Fig. 6C), but the instantaneous current amplitude at +75 mV was unchanged. These observations again imply that
voltage-dependent gating steps are modified by the reaction
with MTS reagents.
In all cases, the effects of MTSES or MTSET were not reversed
after moving the pipette in a solution without MTS reagents but only
after addition of the reducing agent dithiothreitol. This demonstrates
that the substituted thiol side chains react covalently with the
hydrophilic MTS reagents and shows that these residues are in contact
with the cytoplasmic aqueous medium. G188C did not express with
sufficient current density to allow macroscopic recordings from excised
patches. Moreover, in experiments with whole-cell recordings without
modifying agents, we observed in all cells after the initial
establishment of the whole-cell configuration a slow increase of the
current amplitude (i.e. a run-up phenomenon). This feature
prevents the accurate interpretation of experiments performed with MTS
reagents in the pipette solution. We were therefore unable to determine
the accessibility of Cys-188.
The reaction of an MTS reagent with a thiol side chain of a channel
protein occurs in two consecutive steps. The MTS reagent first has to
diffuse from the aqueous solution to the side chain and afterward react
with it in a second step. The rates with which these two reactions
occur cannot be determined independently, but the experimentally
determined reaction rates often give information about the
rate-limiting step in the cascade. For four substituted cysteines
(G190C to E193C), we obtained second-order rate constants for
modification with MTSES and MTSET (Fig. 3B). Rate constants for all reactions are well below the values observed for the
modification of thiols in aqueous solution (21), and this observation
suggests that reagent access from the aqueous solution is the
rate-limiting step. The apparent reaction rates can thus be used to
judge whether the reagent diffuses through an anion-selective or
unselective entry pathway. Because MTSES (anionic) and MTSET (cationic)
have similar molecular diameters, differences in reaction rates of a
given cysteine mutant with these two reagents are most likely caused by
an electrostatic interaction, with regions of the channel protein
determining the selectivity of the pore entrance as well as with the
negatively charged thiolate form of the substituted cysteine with
which the MTS compound reacts (21). To obtain the anion to cation
selectivity of the access pathway, one has to correct for the latter
component. For this reason, the measured rate constant ratio
(kMTSES/kMTSET) is
normalized to the relative reactivity of the two reagents in aqueous
solution (21), and this calculation yields the relative anion to cation
selectivity of the access pathway. For the tested substituted thiols
within P3, we obtain values between 9.4 and 120 (Fig. 3C).
These results demonstrate that parts of P3 are in contact with the
aqueous phase and that the access pathway to thiol side chains within
this region is anion-selective.
To test whether P3 contributes to the formation of an anion-binding
site, we performed chemical modification experiments in the absence and
presence of thiocyanate ions (SCN
). If accessible side
chains within P3 are in contact with permeating anions, one would
expect that the reaction of MTS reagents with substituted cysteines
will be impaired under experimental conditions that favor occupation of
the particular binding site within the pore by anions. Several anions
bind more tightly than chloride to sites within the ClC-1 ionic pore
(8, 10) and effectively block chloride currents. We studied the effect
of partial substitution of Cl
with SCN
on
the reaction of substituted cysteines within P3 using the positively
charged MTSET. If SCN
binds close to the substituted
cysteine, MTSET might be unable to react if the thiol side chain is
shielded by bound SCN
nearby. A decrease of either the
degree of current reduction or the second-order reaction rate constants
would indicate that P3 contributes to an anion-binding site. If MTSET
reacts with a residue located elsewhere within the pore, electrostatic
interactions of its positive charge with the bound anion are expected
to boost the access of MTS reagents and will produce opposite results.
For these experiments, we studied Cys-190 and Cys-193. Fig.
4 shows results for these two P3
cysteines as well as Cys-231 in P1. Several lines of evidence indicate
that Lys-231 in P1 makes direct contact with permeating anions (18),
and Cys-231 was therefore used as a control. Partial substitution of
chloride by SCN
completely abolishes the effects of
Cys-231 modification suggesting that SCN
protects this
thiol group. For the thiol side chains substituted within P3, the
effect of SCN
was clearly distinct. Cys-190 as well as
Cys-193 were modified in the presence of SCN
. For
Cys-190, the extent of current reduction and the reaction rate during
MTS treatment was significantly (p < 0.01) decreased. In contrast, for E193C, neither the reaction rates nor the degree of
current reduction was altered in the presence of SCN.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of partial intra- or extracellular
chloride substitution with thiocyanate. A, time course
of modification of K231C hClC-1 channels by 50 µM MTSET
as determined by whole-cell recordings with standard intracellular
solution. The time dependence of the maximum current amplitude is shown
for two different cells, one in standard external solution ( ) and
the other in a solution containing (in mM) 50 NaSCN, 90 NaCl, 4 KCl,2 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4 ( ). Current maxima are normalized to control values obtained before
MTSET application. B, relative current change for
substituted cysteine G190C, E193C, and K231C by MTSET in standard
solutions (hatched bars) and in solutions in which 50 mM NaCl were substituted with 50 mM NaSCN. The
data were obtained with whole-cell recordings (K231C) or excised
inside-out recordings (G190C and E193C). C, reaction rates
for modification of these cysteines under the conditions in
B.
|
|
SCN
as a weak nucleophile could react with MTS reagents,
reduce their concentration, and thus cause a decreased apparent
reaction rate. Although we cannot exclude that any of the applied MTS
reagents reacted with SCN
, the marked difference of the
SCN
effect on the MTS reaction with different substituted
cysteines demonstrates that this is not the major action of this anion. SCN
completely abolishes the reaction of MTS with K231C;
it reduces the reaction with G190C and leaves the reaction with E193C
unchanged. We conclude that Lys-231 directly contributes to the
formation of an anion-binding site and that Gly-190 is located near an
anion-binding site, but obviously anions do not bind as so close to
this side chain that the MTSET compounds cannot reach the thiol group.
Our results do not support the notion that Glu-193 plays a role in anion binding within the ClC pore.
Functional Interaction of P3 Regions--
By using chemical
modification of homo- and heterodimeric cysteine-substituted channels,
we have recently provided evidence that accessible side chains within
P1 of both subunits of a single channel interact with each other in a
way that would be expected if they jointly form a single vestibule
(24). To test whether such an interaction also takes place between the
two P3 regions, we employed a similar experimental strategy.
We engineered tandem constructs (19) to express a homogenous population
of heterodimeric single cysteine-substituted channels. Fig.
5, A
C, illustrates
representative current recordings from heterodimeric WT-G190C channels
under standard conditions (Fig. 5A), during modification by
MTSES (Fig. 5B) and afterward (Fig. 5C). After
reaching steady-state conditions following MTSES modification, a
substantial current component carried by WT-G190C remains (Fig. 5C). By contrast, modification of the double
cysteine-substituted channel causes an almost complete disappearance of
the typical hClC-1 gating phenotype (Fig. 5D). This outcome
is different from that of experiments with cysteine-substituted
channels within the P1 region (24) where chemical modification of homo-
and heterodimeric channel causes identical functional changes. At first
glance, these results appear to support the idea that each P3 region of
a functional hClC-1 dimeric channel forms an independent ion conduction
pathway. However, two lines of evidence demonstrate that this is not
the case. A comparison of the time course of modification (Fig.
5E) demonstrates that the reaction is significantly slower
in the heterodimeric channel than in the homodimeric double cysteine-substituted channel. This result is in clear contrast to the
predictions of a double-barreled channel for which the rate constants
of modification of a certain cysteine should be unaffected by
substitutions in the other protochannel. Whereas for MTSES modification
there is a significant component of unmodified current, the unblocked
current levels after MTSET modification are indistinguishable for homo-
and heterodimeric channel (Fig. 5F). Fig. 5, F
and G, summarizes the results obtained from several different cells tested separately with MTSES and MTSET. Second order
rate constants are significantly reduced in channels with only one
substituted cysteine (Fig. 5G). Similar results were obtained for I191C homo- and WT-I191C heterodimeric channels (data not
shown).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
MTS modification of homo- and heterodimeric
cysteine-substituted hClC-1 channels. A, excised
inside-out patch clamp recordings from a cell expressing WT-G190C
hClC-1 channels before modification. Voltage steps between 165 and
+75 mV in 60-mV steps were applied from a holding potential of 0 mV.
Each test pulse is followed by a fixed 105-mV pulse. B,
current recordings during modification of the same patch as in
A with 2 mM MTSES. The pulse protocol consists
of consecutive voltage steps to +75 mV followed by a 105-mV test
potential during the modification. C, current recordings
from the same patch after modification. Voltage steps are as described
in A. D, current recordings during modification
of patch from a cell expression G190C homodimeric channels with 2 mM MTSES. Same pulse protocol was used as in B. E, time dependence of the normalized maximum current at 105 mV,
obtained from the recordings shown in B (open
symbol) and in D (filled symbol). For this
graph, the instantaneous current amplitude at 105 mV was taken from
each record and plotted versus the time since the beginning
of the MTS application. F, second order reaction rates; and
G, relative current change for modification of homo- and
heterodimeric channels with MTSET and MTSES.
|
|
Fig. 6 illustrates the modification of
P192C and E193C heterodimeric and homodimeric channels by MTS reagents.
In all cases there are different effects on macroscopic currents
through heterodimeric versus homodimeric channels. MTS
modification of heterodimeric channels is not a simple superposition of
the modification of cysteine-tagged pores with a current component by
WT pores that are not modified. Similar to the other tested cysteines
in the P1 and P3 regions, cysteine modification in one subunit does not cause functional effects that are independent from the other
subunit.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Distinct MTS modification of homo- and
heterodimeric channels. A, P192C; B,
WT-P192C; C, E193C; and D, WT-E193C. Inside-out
patch clamp recordings and patches were held at 0 mV and stimulated
every 5 s to voltage steps to +75 mV followed by 105 mV. Current
responses for different times after MTS applications are superimposed.
For A and B, 2 mM MTSET were applied,
and for C and D, 50 µM MTSES.
|
|
The results obtained for single and double cysteine modification in P3
support the idea that this region in both subunits somehow interacts
with the contralateral corresponding region, similar to that observed
for the P1 region. Nevertheless, the functional effects of Cys-190
modification with MTSES in a heterodimeric channel are remarkably
different from similar results obtained within the P1 region. A
possible interpretation of these differences would be that the portion
of the conduction pathway formed by P3 is wide enough to accommodate a
covalently linked MTSES molecule and still permit ion flux. We tested
this possibility by using different cysteine-specific reagents with
larger substituents.
Dimensions of the hClC-1 Pore--
Fig.
7 shows the results of chemical
modification of four cysteine side chains protruding into the pore of
ClC channels by the cysteine-specific reagents, qBBr and MTS-PTrEA.
MTS-PTrEA is a cubic molecule with a length of 8 Å, and qBBr exhibits
a dimension of more than 6 × 10 × 12Å (25). Two cysteines
are accessible to both reagents (K231C, G190C), E193C is only
accessible to MTS-PTrEA, and H237C cannot be reached by either
compound. As all these side chains are accessible to smaller
cysteine-reactive reagents, the most likely possibility is that these
large agents cannot bind because of steric hindrances. The dimensions
of these agents therefore provide estimates for the proportions of the ionic pore of hClC-1.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 7.
Modification of cysteine-substituted hClC-1
channels with MTS-PTrEA and qBBr. A, relative current
reduction; and B, second order rate constants.
|
|
 |
DISCUSSION |
P3 Is a Pore-lining Segment--
By using a combination of
site-directed mutagenesis, heterologous expression, and chemical
modification, we have demonstrated the following: 1) mutations within
the highly conserved P3 region between transmembrane domains D2 and D3
affect anion selectivity of hClC-1; 2) cysteine substitution
experiments indicate that this segment is accessible to internally
applied hydrophilic MTS reagents; 3) the internal access pathway of MTS
reagents to P3 is anion-selective; and 4) SCN
impairs MTS
modification of a P3 thiol side chain (Cys-190). These results are all
consistent with the idea that P3 forms part of the ion conduction
pathway of the hClC-1 pore.
There are qualitative differences of the results obtained with P3
residues and those with P1 or P2 residues (18). For substituted cysteines within P1, the modification of a single cysteine decreases the current amplitude to zero levels, whereas in WT-G190C heterodimeric channels, modified by MTSES, a substantial amount of residual current
could be observed. Moreover, whereas occupation of anionic binding
sites prevents chemical modification of thiol side chains within P1,
for residues belonging to the P3 region either no effect (E193C) or a
less pronounced effect (G190C) could be observed. These results suggest
that P3 may not contribute to the formation of the narrow part of the
hClC-1 pore and most probably is not part of an anionic binding site. A
likely explanation for these results may be that P3 forms part of a
wide internal vestibule.
Estimating the Dimensions of the ClC Pore--
Studying
interactions with high affinity pore blockers has provided many
insights into the structure of the ion conduction pathway of many ion
channels, most notably for organic compounds (26-28) or peptide
blockers (29-31) with voltage-gated potassium channels. There are
currently no specific blockers for ClC channels precluding employment
of this approach. The use of cysteine-specific reagents has provided an
alternative strategy for gaining insights into the dimensions of the
ion conduction pathway of ClC channels.
Our earlier experiments with internal and external MTSES and MTSET
demonstrated that there is a major constriction between Lys-231 and
His-237, most probably the most narrow part of the ClC channel pore,
and that this constriction is less than 6 Å (18). In agreement with
these findings, experiments with various permeant anions provided
evidence that this narrowing has a diameter between 4 and 6 Å (8, 10).
The use of MTS-PTrEA and qBBr now reveals that the pore diameter is
wider than 10 Å at the level of Lys-231 and G190C and that Glu-193 and
His-237 are located in pore regions that are more restricted. From our
data, it would appear that ClC channels exhibit the same hourglass pore
architecture as other classes of ion channels with a constriction
displaying the major selectivity neighbored on both sides with wide
vestibules. This architecture effectively reduces the passage of the
ion through a low dielectric constant medium and thus significantly
diminishes the resistance of the ion conduction pathway (32).
The result with Cys-231 is surprising given that two Cys-231 residues
in a homodimeric ClC channel are close enough to form disulfide bridges
(24). To account for these disparate observations, we suggest that the
pore wall of ClC channels around position 231 is very flexible.
Flexibility of ionic pores has been proposed to account for a stepwise
dehydration of ions while entering the narrow part of an ion channel or
carrier (33, 34), and experiments with oxidizing agents have recently
directly demonstrated such an elasticity for the outer mouth of sodium
channels (35).
For hClC-1, this interpretation gives a structural basis for two other
previous experimental results. An investigation of the blocking action
of iodide suggested that there are conformational changes of the ionic
pore of hClC-1 (8). A flexible pore region around Lys-231 could account
for this finding because of the great functional importance of this
residue (18). Moreover, SCN
exerts a puzzling effect on
ion conduction through hClC-1. External SCN
blocks
chloride currents in the negative and low positive voltage range, but
at very positive potentials there is an outwardly rectifying current
carried by SCN ions (10). Such a behavior (usually denoted as "punch
through" effect) can be explained with a flexible pore (36, 37).
Pore Stoichiometry of ClC Channels--
In 1982, Miller (38)
suggested a novel and unique pore architecture for the ClC-0 chloride
channel. Based on single channel recordings, he proposed that these
channels exhibit two identical and functionally independent protopores
("double-barreled shotgun" model). In the following years, Miller
and colleagues provided several lines of evidence supporting this
concept (39, 40). Later experiments with heterodimeric ClC-0 channels
in which the primary sequence of one subunit was mutated at two
specific residues (16, 41) demonstrated that the the unitary
conductance as well as the ion selectivity of each subconductance state
is determined by only one subunit supporting Miller's original
proposal. The results of the work contained in the present paper and
those of an earlier study (24) now reveal that two regions that are
critical for the ion conduction in hClC-1 exhibit a behavior that
reveals a functional interaction between the two subunits. These
experiments are inconsistent with the proposed functional
independence of the two protopores and appeared to be more consistent
with a single conduction pathway than with the original double-barreled
shotgun hypothesis. The different outcome of experiments performed with ClC-0 and hClC-1 cannot be explained by isoform-specific differences in
pore stoichiometry. As ClC-0 and hClC-1 share high sequence identity,
corresponding mutations in both isoforms cause similar functional
alterations, and both isoforms display equally spaced subconductance
states (38, 42); there is little doubt that ClC-0 and ClC-1 exhibit an
identical quaternary structure of the pore.
These two experimental results represent a novel and interesting
feature of ClC-type chloride channels. They either exhibit a single
pore in which subconductance states occur that are completely independent, and we have at present only suggestions how this can
happen (9), or they exhibit two ion conduction pathways that are
independent in certain aspects but clearly interact in others. Another
possibility is a combination of both, the existence of one or two
common vestibules connected by two distinct conduction pathways. A
definitive answer to this open question will provide important insights
into the function of ClC-type chloride channels.
Conclusion--
We have identified a segment (P3) that contributes
to the formation of a wide internal vestibule of ClC channels.
Experiments with cysteine-specific reagents of different size revealed
that ClC channels exhibit a pore architecture quite similar to cation channels, with a narrow region that is important for anion-cation selectivity in the center and wide vestibules on both sides. These experiments provide novel insights into the architecture and the function of the pore of ClC-type chloride channels.