From the * Department of Medicine, and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-2372
Voltage-gated Cl channels belonging to the ClC family exhibit unique properties of ion permeation
and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl
channel
(hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or
intracellular I
and related anions. Extracellular and intracellular I
exert blocking actions on hClC-1 currents
that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide
(Br
) and polyatomic (SCN
, NO3
, CH3SO3
) anions. In addition, I
block is accompanied by gating alterations
that differ depending on which side of the membrane the blocker is applied. External I
causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating,
nondeactivating), while internal I
slows deactivation. These different effects on gating properties can be used to
distinguish two functional ion binding sites within the hClC-1 pore. We determined KD values for I
block in three
distinct kinetic states and found that binding of I
to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I
binding suggest that conformational changes affecting the two ion
binding sites occur during gating transitions. These results have implications for understanding mechanisms of
ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.
The molecular cloning of a voltage-gated Cl channel
from the electric organ of Torpedo ( Jentsch et al., 1990
)
and the subsequent characterization of a large number
of mammalian homologs ( Jentsch, 1994
) has established a new gene family (ClC-family) lacking any structural similarity to other known ion channels. At
present, the basic mechanisms responsible for ion permeation and gating in ClC channels are incompletely
understood.
We have focused on the human muscle ClC-isoform,
hClC-1. This dimeric channel (Fahlke et al., 1997b) is
important physiologically for the control of sarcolemmal excitability and it is the genetic locus in mouse,
man, and goat for a specific form of inherited myotonia
(Steinmeyer et al., 1991
; Koch et al., 1992
; George et al., 1993
; Beck et al., 1996
). We have previously characterized a recombinant human ClC-1 (hClC-1) expressed heterologously in both Xenopus oocytes and human embryonic kidney cells (Fahlke et al., 1995
, 1996
),
and found that its functional attributes are identical to
native skeletal muscle channels (Fahlke and Rüdel, 1995
).
Investigation of the dependence of gating properties
on pH and chloride concentrations has helped us to
develop a first gating model of this channel (Fahlke et
al., 1996). Gating of hClC-1 appears to be mediated by two
structurally distinct mechanisms: a fast voltage-dependent
process and a slow voltage-independent process controlling opening and closing transitions through block of the pore by a probable cytoplasmic gate (Fahlke et
al., 1996
). The voltage-dependent process governs the
distribution of open channels in three kinetically distinct states: fast deactivating, slow deactivating, and
nondeactivating. More recently, we have characterized
an hClC-1 mutation (G230E) that causes autosomal dominant myotonia congenita and confers altered ion
selectivity on the channel (Fahlke et al., 1997a
). In that
report, examination of the effect of I
on both wild-type and mutant channels provided preliminary evidence for the existence of two distinct anion binding sites
within the hClC-1 pore.
To improve our understanding of the basic properties of the hClC-1 pore, and to learn more about the relationships between gating and permeation, we now investigate in more detail the interaction of I and other
analogs of the normal permeant ion with these binding sites. One specific goal of this investigation was to determine if the different conducting states defined by
our gating model exhibit differences in ion binding
characteristics. This work reveals clear differences in
the characteristics of I
block exhibited by the three kinetic states and implies that conformational changes
within the conduction pathway are occurring during
gating. We also define the hClC-1 conduction pathway
as a multi-ion pore, and argue in favor of an ion-selectivity mechanism based on differential ion binding.
These results provide much needed characterization of
the ion permeation process and clarify a functional link
between gating and ion conductance in hClC-1.
Oocyte Preparation and Two-Electrode Voltage Clamp
Isolation, maintenance, and cRNA injection of Xenopus oocytes
were performed as previously described (Beck et al., 1996). Standard two-microelectrode voltage clamp was performed using an
amplifier (OC-725B; Warner Instruments Corp., Hamden, CT). Microelectrodes were pulled from borosilicate glass to have a resistance between 0.7 and 1.3 M
when filled with 3 M KCl. The oocytes were bathed in ND-96 solution (Dascal et al., 1986
) containing 96 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 HEPES (adjusted to pH 7.4 with NaOH). To test the effect of various anions on hClC-1 currents, the bathing solution was
changed to a modified ND-96 in which NaCl was replaced by an
equimolar quantity of NaSCN, NaNO3, NaCH3SO3, Na-cyclamate,
or Na-gluconate. For the calculation of relative current amplitudes, both instantaneous and late current amplitudes were divided by the instantaneous current amplitude measured at
145
mV in the same cell using standard ND-96 solution. In general,
endogenous oocyte chloride currents can be distinguished easily
from hClC-1 currents by their different kinetics. Among the various reported types of endogenous oocyte chloride currents, calcium-activated chloride currents bear the closest resemblance to
hClC-1. However, calcium-activated chloride currents display a
clear activating phase upon voltage steps to positive potentials (Tokimasa and North, 1996
) that is absent in hClC-1-expressing cells at similar test potentials (Fahlke et al., 1996
). Therefore, oocytes exhibiting an activating component larger than 1 µA at +55
mV were excluded from analysis.
Whole-Cell Recording
HEK-293 cells (CRL 1573; American Type Culture Collection,
Rockville, MD) were stably transfected by the calcium phosphate precipitation method using the plasmid pRc/CMV-hClC-1 as described (Fahlke et al., 1995).
Standard whole-cell recording (Hamill et al., 1981) was performed using an Axopatch 200A amplifier (Axon Instruments,
Foster City, CA). Pipettes were pulled from borosilicate glass and
had resistances of 0.6-1.0 M
. Cells with peak current amplitudes <10 nA were used for analysis. More than 60% of the series
resistance was compensated by an analog procedure. The calculated voltage error due to series resistance was always <5 mV. No
digital leakage or capacitive current subtraction was used. Currents were low pass filtered with an internal amplifier filter and
digitized with sampling rates at least three times larger than the
filtering frequency using pClamp (Axon Instruments). Cells were
held at 0 mV for at least 15 s between test pulses.
The standard bath solution contained (mM): 140 NaCl, 4 KCl,
2 CaCl2, 1 MgCl2, and HEPES, pH 7.4. In experiments testing the effect of external I or other anions, the standard bath solution
was modified by replacing variable amounts of NaCl with equimolar
quantities of NaI or the corresponding sodium salt of other anions. For determination of I
dissociation constants (KD) for the
extracellular ion binding site, measurements were initially made
in an extracellular solution composed of (mM): 140 Na-gluconate, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, and then changed
to a modified solution in which Na-gluconate was replaced by
NaI. For experiments with I
-containing solutions, agar bridges
(3 M KCl in 0.1% agar) were used to connect the bath solution to
the amplifier.
The standard pipette solution was (mM): 130 NaCl, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.4. In some experiments, CsCl was substituted for NaCl without appreciable differences in the results. For
determination of KD for I binding to the intracellular site, measurements were made in a pipette solution containing (mM): 50 NaCl, 80 Na-gluconate, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.4, or
in solutions in which Na-gluconate was replaced by equimolar
NaI. All solutions were adjusted to pH 7.4 with NaOH or CsOH.
Unless otherwise stated, standard solutions were used. For experiments with I
-containing solutions, agar bridges (3 M KCl in
0.1% agar) placed inside the patch pipette were used to connect
solutions with the amplifier.
The concentration dependence of the reversal potential was determined in stably transfected HEK 293 cells under bi-anionic conditions using three different intracellular solutions (mM): (a) 44 NaI, 5 EGTA, 10 HEPES, 200 sucrose; (b) 88 NaI, 5 EGTA, 10 HEPES, 100 sucrose; (c) 132 NaI, 5 EGTA, 10 HEPES, in combination with three different extracellular solutions (mM): (a) 49 NaCl, 1 CaCl2, 5 HEPES, 200 sucrose; (b) 98 NaCl, 1 CaCl2, 5 HEPES, 100 sucrose; (c) 147 NaCl, 1 CaCl2, 5 HEPES. All solutions were adjusted to pH 7.4 with NaOH. Reversal potentials were measured after a 2-3 min equilibration time period after establishment of the whole-cell configuration.
Excised Patch Recording
For recording from inside-out excised patches (see Fig. 7), pipettes were pulled from borosilicate glass to have resistances between 1.2 and 2.0 M, coated with Sylgard, and filled with standard extracellular solution. The bath solution was identical to the standard intracellular solution described above for whole-cell recording. Using a solution changing system (SF-77 Perfusion Fast-Step System; Warner Instrument Corp.), the intracellular membrane side of the patch was first exposed to a solution containing
(mM): 50 NaCl, 80 Na-gluconate, 2 MgCl2, 5 EGTA, 10 HEPES,
pH 7.4, and baseline recordings were made. Subsequently, the
solution was changed to (mM): 50 NaCl, 50 NaI, 30 Na-gluconate, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.4, and the measurements were repeated.
Data Analysis
Current deactivation was tested after a 50-ms prepulse to +55
mV. The time course of current deactivation was fit with an equation containing a sum of two exponentials and a time-independent value as follows: I(t) = a1exp(t/
1) + a2exp(
t/
2) + d,
where a1, a2, and d are amplitude terms,
1 and
2 are time constants for fast and slow deactivation, respectively. The fractional
current amplitudes were calculated by dividing by the peak current amplitude (Imax) as follows: A1 = a1/Imax, A2 = a2/Imax, C = d/Imax (Fahlke et al., 1996
).
For the calculation of I dissociation constants (KD) for the external binding site, fractional current amplitudes (A1, A2, and C)
determined at several test potentials were plotted versus the extracellular [I
]. The KD values at given test potentials were obtained as described in RESULTS. Calculation of KD for the intracellular binding site was performed by plotting reciprocal values of
the deactivation time constants (
1
1,
2
1) versus the intracellular [I
], and then deriving KD as a fit parameter by the method
explained in RESULTS.
Block of hClC-1 by External I
To examine the effect of external I on hClC-1, initial
experiments were performed in Xenopus oocytes to permit current recording from the same cell in the presence of various external solutions. Fig. 1 illustrates current recordings made in oocytes expressing hClC-1 before (Fig. 1 A) and after (Fig. 1 B) substitution of 96 mM NaCl by equimolar NaI in the extracellular solution. External I
causes a significant reduction in the
`instantaneous' current amplitude measured 2 ms after
hyperpolarizing voltage steps from a holding potential
of
30 mV, and shifts the reversal potential to a more
positive voltage (Fig. 1, C and D), indicating a greater permeability of hClC-1 for Cl
than I
. In addition, external I
affects hClC-1 gating, resulting in less complete deactivation (Fig. 1 B) and loss of the characteristic inverted bell shape of the steady state current-voltage relationship observed at negative test potentials
(Fig. 1 D). Similar effects of external I
on hClC-1 have
been observed in mammalian cells by whole cell recording (Fahlke et al., 1997a
).
The effect of external I on hClC-1 current amplitude is concentration and voltage dependent. Increasing the external I
from 16 to 96 mM causes a concentration-dependent reduction of instantaneous (Fig. 1
C) normalized inward current amplitudes recorded at
voltages negative to the reversal potential. At voltages positive to the reversal potential, reduction of normalized instantaneous outward current amplitude is near
saturation at 16 mM I
. Thus, external I
reduces both
inward and outward current, having its greatest effect
on outward currents measured at positive potentials.
These data are consistent with voltage-dependent block
of hClC-1 by external I
.
Block of hClC-1 by Other Extracellular Anions
We investigated the effect of other extracellular anions
on hClC-1 expressed in oocytes by substituting 48 mM
Cl in the external solution by equimolar amounts of
Br
, SCN
, NO3
, CH3SO3
, cyclamate, or gluconate.
Fig. 2 shows representative voltage-clamp recordings
for control conditions (Fig. 2 A), 48 mM CH3SO3
(Fig.
2 B), 48 mM NO3
(Fig. 2 C), and 48 mM SCN
(Fig. 2
D). The equimolar substitution of Cl
with each of
these anions causes a variable reduction of inward current amplitudes within the negative potential range.
These effects vary depending on the replaced anion
and therefore are not simply caused by reduction of
the external Cl
concentration.
Plots of normalized current amplitudes versus voltage for seven different ionic conditions are shown in
Fig. 3. Fig. 3, A and B shows substitution experiments
with anions believed to be permeant (Br, SCN
,
NO3
), while Fig. 3, C and D illustrate data from experiments in which the substituted anions were suspected
of being impermeant (CH3SO3
, cyclamate, and gluconate). All anion substitutions cause a shift of the
reversal potential to more positive voltages, indicating lower permeability, relative to Cl
, for each of the
tested anions. In addition, normalized inward and outward current amplitudes are blocked by all anions except gluconate (the effect of gluconate on outward current can be explained by the reduction of external
Cl
concentration). Blocking anions also affect gating
properties, as can be observed in the steady state current-voltage plots, especially at voltages negative to the
reversal potential (Fig. 3, B and D). The blocking potency and the potency to change current kinetics are
correlated (Fig. 3 E).
Gating Effects of External I on hClC-1
We next studied in more detail the effect of external I
on hClC-1 gating properties by using whole-cell recording of HEK-293 cells stably expressing the channel
(Fahlke et al., 1996
). Either in the absence or presence
of extracellular I
, the time course of current deactivation upon hyperpolarizing voltage steps can be fit with
a sum of two exponentials and a constant value. These
fits provide two different data sets: the time constants
of deactivation (
1 and
2 for fast and slow deactivation, respectively), and the fractional amplitudes of two
deactivating and one nondeactivating current components. We interpret the fractional current amplitudes
as estimates of the proportion of channels existing in
each of three different kinetic states: fast, slow, and
nondeactivating (Fahlke et al., 1996
).
Fig. 4 illustrates the effect of external I on these gating parameters for hClC-1 stably expressed in HEK-293
cells. Replacement of 40 mM NaCl by an equimolar
concentration of NaI in the external solution has no effect on the time constants of deactivation (Fig. 4 A),
but there are dramatic changes in the voltage dependence of the fractional current amplitudes. In Fig. 4,
B-D, a pronounced concentration-dependent leftward
shift of the fractional amplitudes for fast deactivating
(Fig. 4 B, A1), slow deactivating (Fig. 4 C, A2), and nondeactivating (Fig. 4 D, C) current components can be
seen. In the negative voltage range in which this effect was observed, there is an increase of the fraction of
channels that either deactivate with a slow time constant or do not deactivate at all. This behavior of the
fractional current amplitudes explains the less complete deactivation observed in the presence of external I
(Fig. 1 B).
Kinetic States Differ in Affinity for External I
Because hClC-1 is conducting in all three kinetic states
(Fahlke et al., 1996) and I
binds to a site within the conduction pathway (Fahlke et al., 1997a
), it is reasonable
to hypothesize that I
can bind to the channel whether
it is in the fast, slow, or nondeactivating state (SCHEME I).
(SCHEME I)
In this scheme, the channel can exist in one of three
states, A1 (fast deactivating), A2 (slow deactivating), and
C (nondeactivating) in the absence of I, and similarly
it can exist in one of three states (A1-I
, A2-I
, and C-I
)
when I
is bound. The I
concentration dependence of
the fractional current amplitudes (Fig. 4 A) indicates
that the rate constants connecting the I
-bound states
(A1-I
, A2-I
, and C-I
) are different from those connecting the unbound states (A1, A2, C). At saturating I
concentration, all channels are occupied by I
, and the
measured fractional current amplitudes thus represent the distribution of channels only in the three I
-bound
kinetic states. At all voltages, the measured fractional amplitudes of the fast (A1) and the slow (A2) deactivating component reach limiting values of zero (Fig. 5, A
and B) at high I
concentrations. Correspondingly, the
constant fractional amplitude (C) approaches a value
of one (Fig. 5 C). Therefore, reaction rate constants between the three different I
bound states (A1-I
, A2-I
,
and C-I
) must be negligible, and we can simplify the
state diagram (SCHEME II).
In this scheme, each I bound state can be reached
only from the corresponding unbound state. Transitions between corresponding bound and unbound
states can be characterized by two rate constants: a first
order dissociation constant (koff) and a second order association constant (kon) for I
. The proportion of channels in a particular kinetic state (i) occupied by I
is
given by [I
]/([I
] + KD,i), where KD,i is a dissociation
constant equal to the ratio koff,i/kon,i. This expression assumes that binding of I
to the channel equilibrates
much faster than the voltage-dependent gating process,
and that only one external I
binds to the channel in
each kinetic state. The latter assumption is tested below. The experimentally determined fractional current amplitude for a given kinetic state (Ai) in the presence
of I
is a weighted mixture of two different probabilities: pI(i) for I
occupied channels, and po(i) for unoccupied channels; these variables are related in the following equation:
![]() |
(1) |
![]() |
(2) |
in which i is a given kinetic state (fast, slow, or nondeactivating), A(i) is the probability for a given state at a
specific I concentration and test voltage, while po denotes the probability without and pI with I
bound to
the channel. This probability expression can provide information on the dissociation constant for I
in the
three different kinetic states, and by analyzing data from several test potentials, we can determine the voltage dependence of KD.
This analysis does not exclude the possibility that
more than one external binding site exerting similar effects on gating exist per channel pore. To test this possibility, we determined Hill coefficients (n) from plots
of fractional current amplitude versus [I] by fitting a
modified version of Eq. 2:
![]() |
(3) |
We determined the concentration dependence of
the effect of external I on the three fractional current
amplitudes (Fig. 5). Fractional current amplitudes were
determined from current recordings made during various test pulses between
75 and
165 mV after a maximally activating prepulse (+55 mV) in the presence of
different I
concentrations. To avoid effects caused by
changing concentrations of Cl
, the extracellular Cl
concentration was held constant (10 mM) and bath
gluconate was varied inversely with changes in I
concentration. Gluconate has no effect on gating properties of hClC-1 (Fig. 3).
We evaluated our model in two steps. First, we determined the Hill coefficient for each fractional current
amplitude by fitting regression lines of the log (A/
(Amax A) vs. log([I
]) relationship where A represents the different fractional amplitudes (Fig. 5, A-C,
insets). All calculated slopes had values
1, consistent with a single external I
binding site. Next, we fitted
Eq. 2 to the data (Fig. 5, A-C) to obtain the voltage dependence of the dissociation constants for I
in each
kinetic state and the voltage dependence of po and pI (these fit parameters are plotted in Fig. 6, see below).
For the A1, A2, and C components, the data are well fit
with a single hyperbola, although data for the slow deactivating component (A2) are more scattered than for
the other two components. This suggests that this
model is a reasonable first approximation of the interaction between I
and hClC-1.
This analysis gives us the dissociation constant, KD,
for external I binding to the channel in each kinetic
state (Fig. 6, A and B). For the fast deactivating component (A1), KD is nearly voltage independent. By contrast, for both the slow deactivating component (A2)
and the nondeactivating component, the KD is voltage
dependent and can be well fit with the Woodhull formula (Woodhull, 1973
), giving the KD (0 mV) and the
electrical distance
(Fig. 6, A and B, Table I). Furthermore, fits of the data in Fig. 5 with Eq. 2 provide limiting values of P at very high (pI) (Fig. 6 C), or zero (po)
(Fig. 6 D) I
concentration that can be used to describe
the fractional current amplitudes when all external
binding sites are either occupied or unoccupied by I
.
The plot of the calculated po for the three different kinetic states resembles the experimentally determined
voltage-dependent behavior of the fractional current
amplitudes measured in the absence of I
(Fahlke et
al., 1996
). By contrast, the plot of calculated pI for the
three different states indicates that binding of external I
locks the channel in the nondeactivating state (Fig. 6 C).
Table I.
Parameters for I |
Block of hClC-1 by Internal I
To examine the effect of internal I on hClC-1, we initially recorded currents from inside-out patches excised
from cells stably expressing the channel. Currents were
recorded from the same patch before and after application of 50 mM NaI to the cytoplasmic face of the membrane. In the presence of 50 mM I
, the inward current
amplitude is greatly reduced, whereas the outward current is unchanged (Fig. 7, A and B). In addition to reduction of the inward current amplitude, there is an apparent slowing of the deactivation process (Fig. 7 B), and
the pronounced inward rectification of the instantaneous current-voltage relationship is abolished (Fig. 7 C).
We next examined the concentration dependence of
the effects of internal I on hClC-1 by using whole cell
recording (Fig. 8). Currents recorded from cells exposed to various intracellular I
concentrations were
normalized to levels measured at the most positive test
potential, a valid procedure in view of the demonstrated lack of effect of internal I
on outward current.
Fig. 8, A and B illustrates the concentration-dependent reduction of normalized inward current amplitude by
internal I
. The effects are consistent with intracellular
I
block of hClC-1 currents.
Block of hClC-1 by Other Internal Anions
Other anions exert similar effects on hClC-1 when
present inside the cell. Fig. 9 A shows whole-cell recordings made from cells dialyzed intracellularly with 50 mM NaI (Fig. 9, A and B), 50 mM NaSCN (Fig. 9, C and
D), or 50 mM NaNO3 (Fig. 9, E and F). Compared with
recordings made with standard pipette solutions, the
deactivation process is much slower for all tested anions, but this kinetic effect is most pronounced for I.
Analysis of the voltage dependence of the instantaneous current amplitude (Fig. 9, B, D, and F) shows
that the degree of inward rectification of the instantaneous current amplitude is also decreased. These results indicate that several anions are able to interact with an internal ion binding site. As observed for the
external binding site, the blocking potency of internally applied anions is correlated with the potency to
change current kinetics (Fig. 9 G).
Kinetic Effects of Internal I
We further evaluated the kinetic changes caused by internal I by examining the channel with whole cell recording in the presence of various concentrations of
NaI in the pipette solution. The time course of current
deactivation measured under these conditions could be
well fit with a function consisting of two exponentials and a constant term as described in METHODS. Both fast
and slow deactivation time constants are increased in a
concentration-dependent manner by intracellular I
,
and by contrast to the effect of external I
, both deactivation time constants become voltage dependent (Fig. 10, A and B). Interestingly, however, the two time constants behave in opposite directions in response to voltage. Whereas the fast time constant increases with
more negative test potentials (Fig. 10 A), the slow time
constant decreases with hyperpolarization (Fig. 10 B).
We have previously modeled hClC-1 deactivation as a
first order process mediated by a cytoplasmic gate
(Fahlke et al., 1996). The occurrence of fast deactivating, slow deactivating, and constant current components corresponds with the existence of three populations of channels differing in the affinity of the internal
vestibule of the ionic pore for this blocking particle.
For each kinetic state in the presence of internal I
,
there will be a mixed population of channels whose internal binding site will be occupied or not occupied by
I
(see Scheme 2). Therefore, both deactivation time
constants in the presence of internal I
will be a
weighted mixture resulting from these two channel
populations. By analogy to Eq. 2, we can relate the I
dissociation constant (KD) for the internal binding site
to the deactivation time constants by the following formula:
![]() |
(4) |
where i is either the fast (
1) or slow (
2) deactivation
time constant determined in the presence of internal
I
, and
min,
max, and KD are fit parameters. We restricted this analysis to the fast- and slow-deactivating
components because, in the presence of internal I
, we
are unable to distinguish the constant component from
an incomplete deactivation in the fast or slow mode.
We assumed that intracellular Cl
equilibrates with the
internal binding site much faster than the deactivation
process. We determined KD values for these two kinetic
states at different test potentials (Fig. 11, A and B). The
two kinetic states analyzed in this manner exhibit differences in their affinities for I
and in the voltage dependence of the effect (Fig. 11 C, Table II). Whereas
the fast deactivating state is characterized by a voltage-dependent KD, the values for the slow deactivating component are nearly voltage independent. As expected,
the derived values for
min (Fig. 11 D) closely resemble
the experimentally determined values measured in the
absence of internal I
(see Fig. 10). For both the fast
and slow processes, the fit parameter
max
1 is zero, indicating that a channel internally occupied by I
cannot close.
Table II.
Parameters for I |
Multiple Occupancy of the hClC-1 Conduction Pathway
The experiments described above clearly demonstrate
the distinct effects of external versus internal I on
hClC-1, and suggest the presence of two separate ion
binding sites within the ion conduction pathway of
hClC-1. The binding site accessible to internal I
appears to interact more directly with the channel closing
mechanism, whereas the externally accessible site has
effects on the voltage-dependent distribution of channels in the aforementioned three kinetic states. In our
previously described gating model of hClC-1, the latter observation would fit with an alteration in voltage sensing caused by external I
(Fahlke et al., 1996
).
The evidence suggesting that hClC-1 has two distinct
ion binding sites within its conduction pathway raises
the question of whether these sites can be occupied simultaneously. One approach to address whether hClC-1
has a multi-ion pore is by testing for concentration
dependence of the permeability ratio under biionic conditions (Hille, 1992). Therefore, we measured current reversal potentials with whole-cell patch clamp in
HEK-293 cells stably expressing hClC-1 under conditions in which Cl
was the only extracellular permeant
anion and I
was the only intracellular permeant anion. Measurements made with various concentrations
of Cl
and I
in a fixed ratio revealed concentration dependence of the reversal potential and, by inference, of
the PI/PCl permeability ratio (Fig. 12 A). This finding is
consistent with ion-ion interactions within a multi-ion
pore.
A second line of evidence supporting the idea that
hClC-1 is a multiply occupied pore comes from the
electrical distance as obtained from Woodhull fits to
the voltage dependence of the I dissociation constant
to the slow deactivating component (Table I). This
number is greater than one, a finding typical for multi-ion channels (Hille and Schwarz, 1978
).
Based on results demonstrated for ClC-0 (Pusch et
al., 1995), we also tested for anomalous mole fraction
behavior in hClC-1 expressed in oocytes using mixtures
of Cl
with either I
or SCN
. Fig. 12, B and C shows
plots of normalized peak instantaneous current versus
the mole fraction of the tested anion. In these experiments, we observed no minimum value for normalized
current at any tested mole fraction. We also tested mixtures of Cl
with NO3
and similarly did not observe a
minimum value in current versus mole fraction plots.
The absence of anomalous mole fraction behavior in
hClC-1 does not exclude a multi-ion permeation mechanism (Hille, 1992
).
Functional Alterations of Ion Binding Sites by Voltage-dependent Gating
In this paper, we extend our earlier observations suggesting the existence of two distinct ion binding sites in
the hClC-1 conduction pathway (Fahlke et al., 1997a).
Specifically, we have characterized in more detail the
ability of I
and other anions to block hClC-1 when applied from both sides of the cell membrane. Examining
the effects of external versus internal anion block of
hClC-1 has helped us distinguish two fundamentally different ion-channel interactions by virtue of their distinct effects on the kinetics and voltage dependence of
channel gating. The ion binding sites responsible for
mediating block of Cl
current appear to be identical
to those through which anions exert their effects on
gating based upon the close correlation of the two phenomena (Figs. 3 and 9).
Interactions between blocking anions and the channel pore depend upon the kinetic state, as illustrated by
the effect of voltage-dependent gating events on the
quantitative parameters of external and internal ion
binding (Tables I and II). By examining the I concentration dependence of two parameters, fractional current amplitudes (reflecting external I
effects) and the
deactivation time constants (reflecting internal I
effects), we were able to discern remarkable changes in
the apparent affinity of hClC-1 for I
during voltage-dependent gating transitions. In addition, we observed
significant alterations in the electrical distance of I
binding occurring with changes in the kinetic state.
These state-dependent changes in ion binding reveal
transitional alterations in the interactions between the
open pore and blocking anions as a particular mechanistic feature of voltage-dependent gating in hClC-1.
These observations suggest that voltage-dependent gating events may be accompanied by structural rearrangements within the pore that alter the location of
the ion binding sites within the electrical field and affect ion binding affinity. These data support our previously published hypothesis of voltage-dependent transitions occurring between conducting states in hClC-1
(Fahlke et al., 1996
).
The observed differences in the electrical distances
of the binding sites are quite large, and seem to indicate drastic structural rearrangements of the pore during gating. However, electrical distances are not comparable with physical distances because of the inadequacy of the constant field assumption. In multiply
occupied ion channels (Hille and Schwarz, 1978), measured electrical distances can be much greater than the
physical distances and may even exceed unity. In these
ion channels, even slight physical movements of ion
binding sights can cause large differences in the measured electrical distances.
Relationship between Ion Permeation and Gating in hClC-1
It is very apparent from this study of hClC-1 and previously published work on the Torpedo channel, ClC-0
(Pusch et al., 1995; Chen and Miller, 1996
), that ion
permeation and gating are functionally linked in ClC
channels. At the present time, there are differing opinions regarding the explanation for this functional linkage. In ClC-0, evidence has been presented that translocation of the permeating ion through the conduction
pathway confers the majority of the voltage dependence of activation, and that there is little or no contribution of intrinsic protein charge movement to this process (Chen and Miller, 1996
).
We have presented a different viewpoint on the mechanism of voltage-dependent gating in hClC-1 (Fahlke et
al., 1995, 1996
). Based upon macroscopic analysis of
gating, we have proposed a model in which voltage-
dependent conformational changes modulate gating
by altering the affinity of the channel for a cytoplasmic blocking particle. These voltage-dependent conformational changes result in three different kinetic states in
hClC-1 distinguished by their time course of deactivation. This voltage-responsive phenomenon can be modulated by permeant ions. As presented in this paper, occupation of the external binding site by I
locks the
channel in the nondeactivating state.
Chen and Miller (1996) recently examined the Cl
dependence of ClC-0 activation using measurements of
opening rate constants derived from single channel recording of purified channels reconstituted into planar
lipid bilayers. They found that the external Cl
concentration giving a half-maximal opening rate was not affected by the membrane potential, and this observation
led them to conclude that initial binding of Cl
to the
closed channel is voltage independent. Unlike the closed channel, open ClC-0 channels in lipid bilayers exhibit
two ion binding sites that can sense the membrane potential and are located at electrical distances of 0.35 and 0.65 from the cis side (White and Miller, 1981
). Although it is not entirely clear, it seems logical that the
ion binding site in the closed channel that mediates Cl
activation is the same as the site accessible from the
external solution in the open channel. In hClC-1, it is
interesting to note that the site accessible to the external solution is minimally sensitive to voltage when the
channel exists in the fast deactivating mode (
= 0.1;
Table I), but the same site is located more deeply in the
electric field in both the slow deactivating and constant current conformations.
Mechanism of Ion Selectivity of hClC-1 Channels
The observation that almost every tested anion is permeant and yet capable of blocking the channel from
both sides of the membrane provides information about
the mechanism of ion selectivity in hClC-1. The qualitative similarities of effects of the various tested anions on
current kinetics suggests that all of the anions we tested
interact with the same binding sites. This idea is reinforced by the observed correlation between the potency
to block inward current with the ability to alter macroscopic gating properties (Figs. 3 and 9). The two sites
differ slightly in ionic rank order blocking potency (external site: SCN > I > NO3 > CH3SO3 > Br; internal
site: I > NO3 > SCN). By conventional wisdom, anions that block Cl current do so because of higher affinity
for a binding site within the conduction pathway. For
hClC-1, the permeability sequence determined by examining reversal potentials in the presence of different external anion composition (Cl > SCN > Br > NO3 > I > CH3SO3) (Fahlke et al., 1997a
) correlates inversely with
the blocking potency sequence of the internal site, but
less well with that of the external site. In qualitative
terms, all of the tested anions less permeant than Cl
exert a blocking action. This is consistent with a mechanism of ion selectivity in hClC-1 based on differential
ion binding rather than repulsion or size exclusion.
Binding of ions to sites within the conduction pathway
requires replacement of ion-solvent with ion-channel
interactions (Eisenman and Horn, 1983; Hille, 1992
), a
process in which hydration energy is spent and electrostatic energy is released. The binding of larger (i.e., I
)
or polyatomic (i.e., SCN
, NO3
, CH3SO3
) anions
more tightly than Cl
to the hClC-1 binding sites suggests that hydration forces dominate the ion-channel
interaction consistent with weak binding sites in Eisenman terminology (Wright and Diamond, 1977
; Eisenman and Horn, 1983
). These ion binding sites could
conceivably consist of either a fixed charge with a large
radius, or a weak dipole (Wright and Diamond, 1977
).
The measurement of permeability for different-sized
anions can provide information about the hClC-1 pore
size. Among the larger anions tested, CH3SO3 (ionic diameter 0.50 nm; see Halm and Frizzell, 1992
) can
traverse the pore, whereas gluconate (ionic diameter = 0.59 nm) is impermeant, thus giving an estimate of the
minimum pore diameter between 0.5 and 0.6 nm. The
inability of gluconate to permeate or block the hClC-1
pore suggests that both ion binding sites are located in
a narrow part of the conduction pathway. This is in
clear contrast to voltage-gated sodium and potassium
channels in which a variety of blockers can bind to a
wide vestibule but are impermeant because of size exclusion (Hille, 1992
).
The minimum diameter of the hClC-1 pore appears to
be considerably larger than those found in the conduction pathway of voltage-gated potassium channels, but
smaller than that of the nicotinic acetylcholine receptor.
This estimated minimum pore diameter for hClC-1 is similar to apical membrane Cl channels of secretory epithelial cells (Halm and Frizzell, 1992
) and to the skeletal
muscle calcium channel (McCleskey and Almers, 1985
).
Conclusion
In summary, the experimental data we present here provides new insights into the nature of the ion conduction pathway in hClC-1. Our studies suggest that hClC-1 has a rather wide ionic pore that is multiply occupied, and is functionally characterized by two distinct ion binding sites. Both sites appear to be weak interacting sites in Eisenman terminology, and the mechanism of ion selectivity in hClC-1 involves differential ion binding. Lastly, we provide evidence that the hClC-1 pore is dynamic in that conformational changes within the conduction pathway underlie the functional link between gating and permeation. These results provide a framework for understanding ion permeation in hClC-1 and will facilitate future experiments aimed at defining the structure and function of ClC channels.
Address correspondence to Dr. Christoph Fahlke, Department of Medicine and Pharmacology, Vanderbilt University Medical Center, 21st Avenue South at Garland, Nashville, TN 37232-2372. Fax: 615-343-7156; E-mail: cfahlke{at}mbio.mc.vander bi lt. edu
Received for publication 7 February 1997 and accepted in revised form 26 August 1997.
We are grateful to Dr. Louis DeFelice and Dr. Richard Horn for their critical reviews of the manuscript.
This work was supported by grants from the Muscular Dystrophy Association (A.L. George, Jr. and Ch. Fahlke) and the Lu-cille P. Markey Charitable Trust (A.L. George, Jr.). C. Dürr was on leave of absence from Kreiskrankenhaus Krumbach (Krumbach, Germany). Ch. Fahlke was supported by the Deutsche Forschungsgemeinschaft (DFG, Fa301/1-1), and A.L. George, Jr. is a Lucille P. Markey Scholar.
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