From KU Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, B-3000 Leuven, Belgium
We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl current (ICl,swell) in BC3H1 myoblasts. This Cl
current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl
currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from
swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (~60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10-80 ms) and a voltage-dependent slow time constant (>100
ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration
or reducing the Cl
concentration of the extracellular solution. Replacing extracellular Cl
by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN
> I
> NO3
> Cl
>> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation.
In conclusion, our results show that the voltage-dependent properties of ICl,swell are strongly influenced by external
pH , external divalent cations, and by the nature of the permeant anion.
A chloride current activated by cell swelling (ICl,swell)
has been described in numerous mammalian and nonmammalian cell types (for review, see Nilius et al.,
1996; Strange et al., 1996
). This current is involved in a
number of cellular processes including volume regulation
(Hoffmann and Simonsen, 1989
; Sarkadi and Parker, 1991
; Kubo and Okada, 1992
), transport of organic osmolytes (Kirk et al., 1992
; Strange and Jackson, 1995
),
and possibly cell proliferation (Schlichter et al., 1996
;
Schumacher et al., 1995
; Voets et al., 1995
, 1997
).
ICl,swell in the different cell types shares many properties,
including a I
> Br
> Cl
>> gluconate permeability
sequence, outward rectification, block by NPPB, and a
dependence on intracellular ATP (Nilius et al., 1996
).
In contrast, large differences have been observed in the rate and extent of current inactivation at depolarizing
potentials, which is prominent in many cell types, such
as Xenopus oocytes (Ackerman et al., 1994
), C6 glioma
cells (Jackson and Strange, 1995a
), intestine 407 epithelial cells (Kubo and Okada, 1992
), T84 human colonic cancer cells (Worrell et al., 1989
), M-1 cortical collecting duct cells (Meyer and Korbmacher, 1996
),
and osteoblast (Gosling et al., 1995
), but very weak or
absent in parotid acinar cells (Arreola et al., 1995
), endothelial cells (Nilius et al., 1994
), T lymphocytes
(Schumacher et al., 1995
), and skate hepatocytes (Jackson et al., 1996
). It is unclear whether these differences in voltage-dependent behavior arise from differences at
the level of the channel molecule or from differences
in the cellular environment and/or in the experimental conditions.
In this study, we describe the voltage-dependent properties of ICl,swell in BC3H1 myoblasts. We show that extracellular pH, extracellular Mg2+, the permeant anion, and channel blockers have profound effects on these voltage-dependent properties. These results may help to explain the observed differences between the different cell types and give additional clues to elucidate the molecular nature of the channel proteins underlying ICl,swell.
Culture of BH3H1 Cells
BC3H1 cells (American Type Culture Collection, Rockville, MD) were grown in DMEM containing 10% fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 mg/liter streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 10% CO2 in air. The culture medium was exchanged every 48 h. Cells were detached by exposure to 0.5 g/liter trypsin in a Ca2+- and Mg2+-free solution for ~180 s and reseeded on gelatin-coated cover slips. Cells were used 2-5 d after reseeding.
Solutions
The normal, isotonic bath solution was a modified Krebs solution
containing (mM): 150 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, adjusted to pH 7.4 with NaOH. The osmolality, as measured with a vapor pressure osmometer (Wescor 5500 osmometer; Schlag Instruments, Gladbach, Germany), was 320 ± 5 mOsm. The normal hyposmotic solution contained (mM): 82 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, adjusted
to pH 7.4 with 1 M NaOH. For anion selectivity experiments,
NaCl was replaced by an equimolar amount of NaSCN, NaI,
NaNO3, or Na-gluconate. To evaluate the effect of different external Cl or Mg2+ concentrations, we used a hyposmotic solution containing 90 N-methyl-d-glucamine (NMDG)-Cl,1 10 glucose, and 10 HEPES. External Cl
was then lowered by replacing
NMDG-Cl with mannitol. External Mg2+ was increased by replacing NMDG-Cl with MgCl2 in a way that the Cl
concentration remained unaltered. The osmolality of all hyposmotic solutions was
adjusted to 200 ± 5 mOsm with mannitol. The internal pipette
solution contained (mM): 40 KCl, 100 K+-aspartate, 1 MgCl2, 4 Na2ATP, 0.1 EGTA, 10 HEPES, pH 7.2 with 1 M KOH. Stock solutions of niflumic acid, flufenamic acid (Sigma Chemical Co., St.
Louis, MO) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB;
Research Biochemicals International, Natick, MA) were made in
DMSO (final DMSO concentration
0.1%).
Current Measurements
Currents were monitored with an EPC-7 (List Electronic, Lambrecht/Pfalz, Germany) patch clamp amplifier. Patch electrodes had DC resistances between 2 and 4 M. An Ag-AgCl wire was
used as reference electrode. In Cl
substitution experiments, a
3-M KCl-agar bridge was used and membrane voltages were corrected for liquid junction potentials if necessary. Whole-cell
membrane currents were measured using ruptured patches. Currents were sampled at 1- or 2-ms intervals and filtered at 500 or
200 Hz, respectively. Only patches with series resistances <8 M
were retained and between 70 and 80% of the series resistance
was compensated to minimize voltage errors. Single-channel currents were sampled at 500-µs intervals and filtered at 500 Hz.
Membrane currents under nonswollen conditions were usually
small (<2% of the maximal current during cell swelling, see Fig.
1, A and B) and were not subtracted for analysis. The different
voltage protocols are described in the figures. Unless stated otherwise, the holding potential was
80 mV, and the frequency of
pulse delivery was 0.1 Hz. All experiments were performed at room temperature, between 22 and 25°C.
Data Analysis
Current voltage relations were constructed from the current during a 5-s voltage ramp from 100 to +120 mV, applied every 15 s.
The time course of activation of ICl,swell was constructed by measuring the average current in a small voltage window around +60
mV in successive traces.
The current traces during voltage steps were fit by a biexponential function:
![]() |
(1) |
where I is current amplitude, Afast and Aslow are amplitudes corresponding to the time constants fast and
slow, and Ass is the steady
state component. The first 3 ms after onset of a voltage step were
excluded from the fit.
To obtain the voltage dependence of inactivation, we applied
prepulses to different voltages and measured the instantaneous current amplitude during a step to a fixed potential of +120 mV. These values, normalized to that after a 1-s test pulse to 120 mV, were plotted as a function of the prepulse potential. This plot yields the voltage dependence of the "apparent" open probability (apparent Popen) at the end of the prepulse, assuming that
Popen equals 1 after 1 s at
120 mV. The cells did not tolerate depolarizing prepulses of several seconds, which were required to
obtain a complete steady state. We have therefore used the noninactivating current component Ass, as obtained from the fits to
Eq. 1 and normalized to Ipeak as a measure of the steady state
Popen. Plots of the steady state Popen versus potential were fitted by
a Boltzmann function:
![]() |
(2) |
where V1/2 is the membrane potential for half maximal inactivation, kV is the slope factor, and Pmin is a voltage-independent constant. Afast/Ipeak and Aslow/Ipeak were calculated to estimate the contribution of the fast and slowly inactivating components.
We used "triple pulse" voltage protocols to obtain the voltage dependence of the recovery from inactivation. The first voltage step, a 1-s step to +120 mV, induced current inactivation. Recovery from inactivation during the second voltage step of variable length and to various voltages, was assessed from the current during a third voltage step to +120 mV. The normalized instantaneous current amplitude at +120 mV plotted versus the preceding potential yields the voltage dependence of the recovery from inactivation, expressed as percent. Dose-response curves were fitted with a Hill equation:
![]() |
(3) |
where C is concentration, EC50 is the concentration for half-maximal effect, and n is the Hill coefficient.
Relative ion permeabilities (PX/PCl) were calculated from the shifts in reversal potential induced by the anion substitution using a modified Goldman-Hodgkin-Katz equation:
![]() |
(4) |
where Erev is the shift of the reversal potential, [Cl]n and [Cl]s
are the extracellular Cl
concentrations in the normal and anion
substituted hyposmotic solutions, and [X]s is the concentration of
the substituting anion X. R, T, and F have their usual meanings.
Rate constants were fitted according to Eyring's absolute reaction rate theory, using the equation
![]() |
(5) |
where kV is the rate constant, V is voltage, ko is the rate constant at
0 mV and A determines the voltage sensitivity (see Jackson and
Strange, 1995a).
Fitting of the data with the different models was done using home-written software. The whole cell current can be calculated as I(t) = N · i · Popen(t), with N the number of channels and i the single channel conductance. The differential equations describing the kinetic model were solved analytically for each set of rate constants, and Popen(t) was calculated. The product N · i was then obtained from a linear regression of the measured current I(t) as a function of Popen(t). The simplex method was used to determine a set of rate constants that minimizes the residual sum of squares of the linear regression.
For the simulation of current traces, Popen(t) was analytically calculated using the Mathcad plus 6.0 software (Mathsoft, Inc., Cambridge, MA), and values for N · i at the different potentials were estimated from whole-cell data.
All data points represent mean values ± SEM from at least four different cells.
Cell-swelling Activates a Chloride Current, ICl,swell
Superfusion of BC3H1 cells with the normal hyposmotic solution caused cell swelling accompanied by a
large increase in membrane currents in all the tested
cells (Fig. 1 A). Membrane currents returned to basal
level after switching back to isosmotic solution (Voets
et al., 1997). Current-voltage relations reconstructed from voltage ramps applied during the hyposmotic
challenge (Fig. 1 B) show that the swelling-activated
current reverses close to ECl (
20 mV). We have previously shown that this current is a Cl
current, ICl,swell,
with an I
> Br
> Cl
>> gluconate anion permeability sequence and that it is efficiently blocked by niflumic acid, flufenamic acid, and NPPB (Voets et al.,
1997
). This current inactivates during voltage steps to
positive potentials >+40 mV (Fig. 1 C). The negative
slope of the current-voltage relation seen at very positive potentials (Fig. 1 B) is due to inactivation of the
current during the slow voltage ramp.
We observed single-channel Cl currents in outside-out patches pulled from BC3H1 cells in which ICl,swell
had been previously activated in the whole-cell configuration. Successful patches contained either no (n = 15)
or multiple (3-8; n = 17) active channels. This might
indicate that these Cl
channels are clustered, as has
been proposed by others (Jackson and Strange, 1995b
),
or alternatively that some unknown compound needs
to be present in the outside-out patch to have channel
activity. Fig. 1 D shows some typical single-channel measurements during a voltage step to +120 mV. This outside-out patch initially contained three active channels
(Fig. 1 D), (upper trace), but the number of active channels decreased during the experiment and activity vanished after 45 s. The ensemble current (Fig. 1 E) inactivates with a time course similar to that of the current
measured in whole-cell mode (Fig. 1 C). The amplitude
histogram shows four distinct current levels with a
closed peak at 0.0 pA, and open peaks at 4.9, 10.0, and
15.1 pA (Fig. 1 F), indicating a single-channel amplitude of 5 pA at +120 mV. Single-channel currents
could be measured during voltage steps to potentials
between +60 and +140 mV, and the single-channel
amplitudes at these potentials are summarized in Fig. 1
G. The slope conductance, obtained from a linear fit
through the data points, was 47 pS, which is close to values obtained in cortical collecting duct cells (55 pS;
Meyer and Korbmacher, 1996
), C6 glioma cells (57.2 pS; Jackson and Strange, 1995b
), and T84 epithelial cells
(50-60 pS; Worrell et al., 1989
). A more thorough kinetic analysis of single-channel currents was not feasible due to the rapid loss of channel activity in the outside-out patches.
Characterization of the Voltage-dependent Properties of ICl,swell
The voltage-dependent properties of ICl,swell were studied after a stable current level was reached, which usually required a hyposmotic challenge of ~5 min (Fig. 1
A). Fig. 2 A (inset) shows the protocol that was used to
study the depolarization-induced inactivation of the
current. From a holding potential of 80 mV, 1-s lasting steps to potentials between +30 and +140 mV were
applied, followed by a short step to +120 mV. Current
inactivation occurred at potentials >+40 mV (Fig. 2
A). Both rate and extent of inactivation increased at
more positive potentials. As illustrated in Fig. 2 B, the
time course of inactivation was better fitted by a double than by a single exponential. Fitting was not further improved by including a third exponential term, even
when pulses of longer duration were fitted (not shown).
The time constants of the double exponential fits at
various potentials are depicted in Fig. 2 C. The fast time
constant,
fast, was only weakly voltage dependent, whereas
slow clearly decreased with stronger depolarizations. The contribution of the fast component increased monotonously (Fig. 2 D) with depolarization,
whereas the contribution of the slow component first
increased, and then decreased (Fig. 2 E, bell shape).
The contribution of the steady state component Ass, which was used to estimate the steady state Popen, was
well fitted by a Boltzmann equation with values for V1/2
of +75 mV and for kV of 14.1 mV (Fig. 2 F). It can be
seen that this steady state open probability deviates significantly from the apparent Popen at the end of the 1-s
voltage steps.
Recovery from inactivation was studied using a protocol in which a 1-s voltage step to 120 mV and a second
50-ms voltage step to the same potential were separated
by voltage steps to 0, 40,
80, or
120 mV with variable duration (tv; 10-1,000 ms). As illustrated in Fig. 3
A, the peak current during the second step to +120 mV gradually increased, eventually reaching the same peak
level as during the first step. The recovery of the current as a function of pulse duration tv and voltage is
shown in Fig. 3 B. It is clear that the recovery is significantly accelerated at more negative potentials.
This voltage dependency of the current recovery was
also studied with the protocol shown in Fig. 4 A. After a
1-s voltage step to 120 mV, the membrane potential was
stepped to potentials between 120 and +20 mV for
500 ms, followed by a 50-ms voltage step to +120 mV. It
can be clearly seen that the inward current at potentials
more negative than the reversal potential for chloride (ECl =
20 mV) rapidly increases, indicating that inactivated channels reopen at these potentials. Fig. 4 B
shows the fraction of the current that has recovered at
the end of the 500-ms step as a function of potential.
The time course of the currents at potentials (
40
mV was better fitted by a double than by a single exponential (Fig. 4 C). Both
slow and
fast were voltage dependent and decreased with more hyperpolarized potentials (Fig. 4 D). Similar values for
slow and
fast were
obtained by fitting the data in Fig. 3 B with Eq. 1. Additionally, the relative contributions of the fast and the
slow components were voltage dependent, as the contribution of the fast component increased with hyperpolarization (Fig. 4 E).
It has been recently proposed that ICl,swell undergoes
steady state inactivation in the negative potential range
(V1/2 at 72 mV), similar to the behavior of voltage-gated Na+ and K+ channels (Braun and Schulman,
1996
). Using a similar prepulse protocol as Braun and
Schulman (1996)
, we could observe a decrease in the
peak outward current during a voltage step to +120
mV after a prepulse to voltages between
120 and
30
mV (Fig. 5, A and C). Such a decrease of the peak outward current does not, however, necessarily imply a
steady state inactivation, as it could also be due to an incomplete recovery of the current during the interpulse interval at holding potential
30 mV. Indeed, when
the holding potential between the pulses was changed
to
100 mV, allowing a complete recovery from inactivation, the peak outward current at +120 mV was no
longer influenced by the preceding potential, even for
6× longer prepulses (Fig. 5 B and C). We therefore
conclude that, at least in BC3H1 cells, steady state inactivation in the negative potential range is not present.
Evaluation of Kinetic Models
Jackson and Strange (1995a) proposed a simple linear
model to describe the voltage-dependent behavior of
ICl,swell in C6 glioma cells. They could simulate the observed whole-cell data by manually adjusting the kinetic
parameters of a model with one open and three inactivated states. However, their model predicts a complete inactivation of the current at positive potentials, which
is not completely in agreement with their and our experimental data. We were interested to know whether
such a simple linear model could describe the voltage-dependent behavior of ICl,swell in BC3H1 cells and could
be used to quantify the effects of modulators on this
voltage dependence. We therefore evaluated linear
models with one open and two and three closed states
by fitting these models to both inactivating and recovering currents. The model with two closed states provided good fits to the inactivation of ICl,swell at potentials
80 mV, but the fits to the recovery from inactivation
at potentials
60 mV were relatively poor (Fig. 6 A).
Better results were obtained using the model with one
open and three closed states (Jackson and Strange,
1995a
), which provided good fits to both inactivation
and recovery of ICl,swell (Fig. 6 B). The rate constants for
this model could be reasonably well fitted with Eq. 5
(Fig. 6, C-E), which yielded the parameters of the kinetic model listed in Table I. These parameters were subsequently used to simulate currents traces, which
were compared with the experimental data.
Table I. Parameters of the Kinetic Model |
Simulated traces during 1-s voltage steps from 80
mV to potentials ranging from
120 to +120 mV (Fig.
7 A) strongly resembled the experimental current data
(Fig. 7 B; average current from five cells), with similar
time courses of current inactivation at positive potentials. The predicted open probabilities at the end of the
1-s voltage steps were also relatively well in agreement with the experimental data, although the model predicts a somewhat lower open probability at the end of
the steps to potentials above +120 mV (Fig. 7 C).
Simulated traces using the voltage protocol from Fig. 4
A are shown in Fig. 7 D, along with the experimental data
(Fig. 7 E; average current from five cells). It can be clearly
seen that, especially at potentials less negative than 80
mV, the current recovers from inactivation to a much
lesser extent than predicted by the model. For better
comparison, the predicted and experimental recovery
from inactivation after 500 ms are summarized in Fig. 7 F.
These results indicate that the linear model with one
open and three closed states (Jackson and Strange,
1995a) provides a good description of the inactivation
at positive potentials and of the recovery from inactivation at sufficiently negative potentials, but obviously
fails to describe the recovery from inactivation over the
whole voltage range. We therefore preferred to use the double exponential fitting instead of this kinetic model
to describe the effects of modulators on the voltage-
dependent properties of ICl,swell.
Modulation of the Voltage-dependent Properties of ICl,swell
Effect of pH.We examined the effect of extracellular pH
on the inactivation and recovery of ICl,swell. Fig. 8 A
shows current traces in response to 1-s voltage steps to
+120 mV in hyposmotic solutions at pH 6.4, 7.4, and
8.4. The effect of pH on the peak current amplitude is
marginal, but lower pH values clearly accelerate the depolarization-induced inactivation. Whereas fast was not significantly altered (data not shown),
slow was clearly
decreased at lower pH (Fig. 8 B). Lower pH values
caused a leftward shift of the inactivation curves and a
decrease of the slope factor kV (Fig. 8 C). Additionally,
the contribution of the fast inactivating component to
the total current was increased at lower pH (Fig. 8 D). In
contrast, the rate of recovery from inactivation was apparently not affected by external pH (Fig. 8, E and F).
Effect of Mg2+ and Ca2+.
Extracellular Mg2+ had a similar effect on the voltage-dependent kinetics as extracellular protons. Fig. 9 A shows currents measured in
response to 1-s voltage steps to +120 mV in hyposmotic solutions with different Mg2+ concentrations. High
Mg2+ concentrations clearly accelerate the depolarization-induced inactivation, with a similar decrease of
slow as with lower pH values (data not shown). A dose-
response curve for the inactivation, measured after 1 s
at +120 mV is presented in Fig. 9 B. The concentration for half-maximal effect, obtained by fitting the data
with Eq. 3 was at 2.2 mM. Increasing external Mg2+
concentration from 0.5 to 20 mM caused a leftward
shift of the inactivation curve and a decrease of kV and
Pmin (Fig. 9 C). As for lower pH values, higher Mg2+
concentrations increased the contribution of the fast
inactivating component (Fig. 9 D). The rate of recovery
from inactivation was again not significantly affected
(Fig. 9, E and F). Extracellular Ca2+ also affected the inactivation of the current, albeit with a lower potency.
During a 1-s step to +120 mV, the current inactivated to 39 ± 5% of the peak value with 10 mM external
Ca2+, compared with 24 ± 3% with 10 mM external
Mg2+. No significant changes in the kinetics of the current were observed when Mg2+ in the internal solution
was either removed or increased to 5 mM, indicating
that Mg2+ acts from the outside.
Effect of external Cl
We examined the effect of different external Cl concentrations
on the voltage-dependent properties of ICl,swell. As expected for a Cl
-selective current, lowering the external
Cl
concentration resulted in a decrease of the outward
current and a rightward shift of the reversal potential.
Lowering external Cl
also accelerated the depolarization-induced inactivation. In Fig. 10 A, current traces are
shown in response to a 1-s step to 120 mV with different
external Cl
concentrations. For a better comparison,
traces were scaled to an identical current level at the beginning of the step. Inactivation is clearly accelerated at
lower Cl
concentrations. Whereas
fast was not significantly altered (data not shown),
slow was clearly decreased at lower Cl
concentrations (Fig. 10 B). Lowering
external Cl
caused a leftward shift of the inactivation
curve (Fig. 10 C) and an increase of the contribution of
the fast inactivating component (Fig. 10 D). Furthermore, the rate of recovery from inactivation was clearly
decreased with lower external Cl
concentrations, as can
be deducted from the slower time constants (Fig. 10 E)
and from the recovery after 500 ms (Fig. 10 F).
Swelling-activated Cl channels are not only permeable for Cl
, but also for a number of other inorganic
and organic anions. We therefore investigated the effects of a number of anions (with different permeabilities as compared with Cl
) on the voltage-dependent
properties of ICl,swell. Fig. 11 A gives the relative permeabilities of the tested anions, as calculated from the
shifts in the reversal potential according to Eq. 4. Fig.
11 B shows current traces in response to 1-s steps to
+120 mV with three different external anions, rescaled
as in Fig. 10 B. With the less permeant anion gluconate
as major charge carrier, inactivation is clearly faster
than with Cl
, while with the more permeant anion
SCN
the inactivation is significantly slower. In general,
the permeant anions shift the inactivation curve in parallel with their permeability (Fig. 11 C). With the most
permeant anions (I
and SCN
) as major charge carriers, the contribution of the fast inactivating component
becomes almost negligible, resulting in a quasi monoexponential inactivation behavior. On the other hand,
external gluconate causes an important increase in the
contribution of the fast inactivating component (Fig.
11 D). Furthermore, the rate of recovery from inactivation clearly increased with external gluconate, as compared with Cl
and SCN
(Fig. 11, E and F).
Effect of channel blockers.
Various drugs are known to
inhibit ICl,swell. Fig. 12 A shows current traces in response to a 1-s step to +120 mV before and during superfusion with a solution containing 250 µM niflumic acid. Niflumic acid not only blocks ICl,swell in a voltage-independent manner (Voets et al., 1997), but also accelerates the depolarization-induced inactivation (Fig.
12 B). The channel blockade caused a leftward shift of
the inactivation curve and an increase in the contribution of the fast inactivating component (Fig. 12, C and
D). Furthermore, the rate of recovery was slower in the
presence of channel blockers (Fig. 12, E and F). Similar
effects on the inactivation of the current were obtained
with two other channel blockers, flufenamic acid and
NPPB (data not shown).
Voltage-dependent Properties of the Swelling-activated Cl
Current in BC3H1 Cells
Swelling-activated Cl currents with similar permeation
properties and pharmacology have been described in
numerous cell types (Nilius et al., 1996
; Strange et al.,
1996
). In this study, we focused on the voltage-dependent properties of ICl,swell in BC3H1 myoblasts. A typical
characteristic of this current is its time-dependent inactivation during depolarizing voltage steps. This has
been observed in many other cells, although the voltage sensitivity of the inactivation process seems to vary
largely between different cell types. Reported values for
the midpoint of inactivation are scattered between +40
mV for T84 epithelial cells (Braun and Schulman, 1996
), and +105 mV for myeloma cells (Levitan and
Garber, 1995
), whereas in other cell types inactivation
of ICl,swell seems to be very weak or completely absent.
The variability in the composition of the hyposmotic
solutions used in the different studies on ICl,swell may explain some of the differences in voltage-dependent behavior. Standard hyposmotic solutions used in the different studies differ significantly in their content of
Mg2+ (between 0 and 5 mM) and Cl
(between 55 and
170 mM), which, as shown in this work, can have profound effects on the inactivation of ICl,swell. It is also possible that cells with only weak inactivation of ICl,swell contain Cl
channels with a much lower sensitivity to the
different channel modulators. We could indeed show
that ICl,swell in calf pulmonary endothelial cells inactivates to a similar extent as ICl,swell in BC3H1 cells when
the extracellular Mg2+ concentration was increased to
30 or 60 mM (Voets, Droogmans and Nilius, unpublished observation). This indicates that the Mg2+ sensitivity of the Cl
channels in endothelial cells is at least
one order of magnitude lower than in BC3H1 cells.
Modulation by Extracellular pH and Divalent Cations
Low extracellular pH has been shown to accelerate the
inactivation of ICl,swell in Xenopus oocytes (Ackerman et al.,
1994; Voets et al., 1996
), and C6 glioma cells (Jackson
and Strange, 1995a
), but did not affect the kinetic properties of this current in M-1 cortical collecting duct cells
(Meyer and Korbmacher, 1996
) and parotid acinar cells
(Arreola et al., 1995
). In BC3H1 cells, extracellular pH
strongly influenced the kinetic behavior of ICl,swell without
significantly affecting the instantaneous current amplitude. The inactivation curve was shifted to less positive
voltage and
slow decreased when the extracellular pH was
lowered from 8.4 to 6.4. In contrast, recovery from inactivation seemed to be unaffected by these pH changes.
Increasing the extracellular Mg2+ and/or Ca2+ concentration had a comparable effect on the current inactivation as lowering the pH. Similar results obtained in
cervical carcinoma cells led to the hypothesis that the influx of Cl anions at positive membrane potentials
forces the Mg2+ ions and/or protons into a blocking site
within the channel pore (Anderson et al., 1995
). However, lowering the external Cl
concentration and hence
the influx of Cl
anions did not cause the expected decrease in inactivation, but, on the contrary, resulted in a
faster and more pronounced inactivation. It seems very
unlikely that under these conditions Mg2+ ions can
reach a blocking site within the channel, against the large electrical field. We therefore propose that extracellular Mg2+ ions and possibly also protons bind to an extracellular site, likely outside the electrical field, and that
this binding enhances the inactivation of the channel.
Modulation by the Permeant Anion and by Channel Blockers
An important finding is that the voltage-dependent
properties are dependent on the permeant anion itself.
Indeed, lowering the extracellular Cl concentration
caused a shift of the inactivation curve to less positive
potentials and of the recovery from inactivation to
more negative potentials. Moreover, replacing extracellular Cl
with gluconate, NO3
, I
, or SCN
caused a
shift of the inactivation curve, parallel with the relative permeability of these different anions. The recovery
process was also favored when extracellular anions with
a higher permeability were present. Similarly, the rate
of inactivation of ICl,swell in M-1 cortical collecting duct
cells was shown to decrease when extracellular Cl
was
replaced by SCN
, I
, or Br
(Meyer and Korbmacher,
1996
). These findings indicate that binding of an extracellular anion to the channel, possibly to a site within the ion-selective channel pore, prevents the channel
from closing at depolarizing potentials and favors the
reopening at more negative potentials. The observed
modulation of the voltage-dependent properties by
blockers of ICl,swell (Gosling et al., 1996
) may then result from the fact that these blockers hinder the entering of
the anions into the pore.
Comparison with Cloned Cl Channels
The molecular nature of the channel that underlies
ICl,swell is still far from being resolved (Nilius et al.,
1996). Based on several phenotypical similarities between ICl,swell and the chloride current induced by overexpression in Xenopus oocytes of pICln (Paulmichl et al.,
1992
), ICl,swell was proposed to be mediated by a porin-like channel consisting of pICln molecules (Gschwentner et al., 1995
; Jackson and Strange, 1995a
, 1995b
; Strange et al., 1996
). This hypothesis is, however, incompatible with our recent findings that the pICln-induced
current is endogenous to Xenopus oocytes but different
from ICl,swell (Buyse et al., 1997
; Voets et al., 1996
).
Some of the properties of ICl,swell described in this
work are reminiscent of the characteristics of voltage-gated chloride channels belonging to the ClC family
(Jentsch et al., 1995). A first point of similarity is the effect of the permeant anion on the kinetic properties.
Modulation of the gating of ClC-0 (the major Cl
channel from the Torpedo electric organ) and ClC-1 (the major skeletal muscle Cl
channel) by external Cl
and
other permeant anions has been extensively studied
(Pusch et al., 1995
; Chen and Miller, 1996
; Fahlke et
al., 1996
; Rychkov et al., 1996
). It was proposed that Cl
ions can bind to an anion binding site accessible only
from the exterior, and that this binding and possibly
the movement of the bound Cl
ion within the pore
causes the voltage dependency of the gating (Chen and
Miller, 1996
; Pusch et al., 1995
). The third member of
the ClC family, the ubiquitously expressed ClC-2, was
shown to be activated by cell swelling (Gründer et al.,
1992
; Thiemann et al., 1992
). Additionally, the gating
of ICl,swell and of ClC-0, ClC-1, and ClC-2 is modulated by
external pH (Hanke and Miller, 1983
; Fahlke et al.,
1996
; Rychkov et al., 1996
; Jordt and Jentsch, 1997
).
Other biophysical properties of these ClC channels (inward rectification, Cl
> Br
> I
permeability sequence, single-channel conductances below 10 pS) are,
however, incompatible with those of ICl,swell described in this work. It can, however, not be excluded that ICl,swell is mediated by another (not yet cloned) member of the
still expanding ClC family.
Address correspondence to Bernd Nilius, Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium. Fax: 0032-16-34 5991; E-mail: bernd.nilius{at}med.kuleuven.ac.be
.
We thank Drs. J. Eggermont, V. Manolopoulos, and F. Viana for their helpful comments.
This work was supported by the European grant BMH4-CT96-0602 (coordinator B. Nilius).
NMDG, N-methyl-d-glucamine.
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