From the Department of Physiology, Tokyo Medical and Dental University School of Medicine, Tokyo 113, Japan
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ABSTRACT |
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Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied
in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as
Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum
open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This
suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (>100 µM) produced fast and slow blockade both concentration and voltage dependently. The reduction
in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated
the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the
blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were
58-209 mM, 0.69-0.75, 0.45-0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted
~10-200 ms and could be fitted with single-exponential curves (time constant, l
s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time
constant,
b). A significant decrease in
b and no large changes in
l
s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions
bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+
blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36-150 mM and
1-1.8, respectively (n = 3).
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INTRODUCTION |
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Various kinds of cellular processes that are mediated by
Ca2+ under ordinary conditions can also be activated by
Sr2+. A classic example is the generation of calcium action potential by Sr2+ (Fatt and Ginsborg, 1958). Other
examples are transmitter release at a neuromuscular
junction (Dodge et al., 1969
), uncoupling of electrotonic synapses (Baux et al., 1978
), and maintenance of mechano-electrical transduction in hair cells (Ohmori,
1985
). Activation of Ca2+-activated K+ channels is also
known to be produced by Sr2+ (McManus and Magleby,
1984
; Sugihara and Furukawa, 1986
; Oberhauser et al.,
1988
; Yoshida et al., 1991
).
The biophysical properties of large-conductance Ca2+-activated K+ channels (BK channels)1 have been extensively studied in cultured rat skeletal muscle (Barrett et
al., 1982; Magleby and Pallotta, 1983a
; Ferguson, 1991
)
and in mammalian skeletal muscle T-tubule membrane
incorporated into lipid bilayers (Moczydlowski and
Latorre, 1983
; Vergara and Latorre, 1983
; Oberhauser
et al., 1988
). Similar BK channels have been studied in
many types of cells (Marty, 1981
; Benham et al., 1985
;
Lang and Ritchie, 1990
; Sugihara, 1994
; reviewed by
Latorre et al., 1989
). Two different blocking effects
produced by divalent cations have been reported in BK
channels. One is fast blockade, which appears as a reduction in the amplitude of single-channel current,
and has been studied in detail for Mg2+ (Ferguson,
1991
). The other is slow blockade, which appears as the
production of long shut events, and has been extensively studied for Ba2+ (Vergara and Latorre, 1983
; Benham et al., 1985
; Miller et al., 1987
; Diaz et al., 1996
;
Neyton, 1996
). Fast blockade is also produced by other
divalent cations such as Ca2+, Sr2+, and Ni2+ (Marty,
1981
; Oberhauser et al., 1988
; Ferguson, 1991
). Slow blockade is relatively specific to Ba2+, although it has
been reported that Sr2+, Pb2+, and Cd2+ may also produce slow blockade (Oberhauser et al., 1988
). These previous findings suggest that Sr2+ is a rather special
ion for BK channels in that it can produce activation,
fast blockade, and slow blockade.
The purpose of this study was to investigate the properties of BK channels of goldfish hair cells, particularly
with regard to activation and blockade by analyzing the
effects produced by Sr2+. Regarding general characteristics, such as unitary conductance and Ca2+ and voltage sensitivity, BK channels in goldfish hair cells are largely similar to those in many other preparations, although they do have some unique characteristics, such
as short open times, low sensitivity to Ca2+, and variation in conductance and gating kinetics among channels (Sugihara, 1994). Comparison of the effects of
Sr2+ and Ca2+ on activation contributed to understanding of the activation mechanism. The fast and slow
blockades produced by Sr2+ were then analyzed in detail. The effects of Ba2+ were also analyzed for comparison with Sr2+-induced slow blockade. Slow blockade
produced by Sr2+ was similar to but simpler than that
produced by Ba2+, and thus the former could be useful
for understanding blockade and permeation in BK
channels.
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MATERIALS AND METHODS |
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Preparation and Experiments
The methods for dissociating single hair cells from the inner ear
of goldfish, Carassius auratus, and for the setup of patch-clamp experiments (Hamill et al., 1981) have been described previously (Sugihara and Furukawa, 1989
, 1995
, 1996
; Sugihara, 1994
).
Goldfish were anaesthetized with an intramuscular injection of
ketamine hydrochloride (1 mg/g body weight
1). The dorsal
skull and brain were removed to excise the inner ears. Hair cells
were isolated from the saccular maculae. All experiments were
performed at room temperature (19-22°C). The recording
chamber was first filled with normal Ringer's solution containing
(mM) 120 NaCl, 2 KCl, 2 CaCl2, 5 Hepes, 1.5 NaOH, pH 7.2. When the patch pipette was placed in the bath, a small constant voltage was applied to the pipette to obtain zero current. After the formation of a gigaohm seal in the basolateral surface of the
cell, the pipette voltage was shifted by the value of the liquid
junctional voltage (
4 mV KCl solution [see below] in the pipette against normal Ringer's solution bath), which should hereafter be absent. The bath of normal Ringer's solution was then
replaced with 125 mM KCl solution, and the cell, except for the
membrane patch, was destroyed by being hit with a small air bubble to form an inside-out patch (Sugihara, 1994
). To identify BK
channels, membrane current was monitored while the bath solution was changed from Ca2+-free KCl to KCl with an internal calcium concentration ([Ca2+]i) of 5 µM and to NaCl (125 mM)
with 5 µM [Ca2+]i, and then back to KCl. Experiments were performed with membrane patches in which one or a few BK channels (>150 pS) were detectable. Membrane patches in which a
single BK channel was present were used in experiments to measure open and shut times or reductions in the unitary amplitude.
The seal resistance of the membrane patch was >10 G
.
Data Analysis
Data acquisition and analysis were facilitated by the use of various
custom programs developed in the laboratory. The output of the
patch-clamp amplifier (10-kHz three-pole low-pass filter, EPC7;
List Electronic, Darmstadt, Germany) was recorded with a PCM
data recorder (PR-880; NF Electronic Instruments, Yokohama, Japan) at a sampling interval of 17.4 µs. The replayed data or the
direct output of the amplifier was then passed through a low pass
four-pole Bessel filter (DT-408; NF Electronic Instruments), digitized, and stored on the hard disk of a microcomputer (N10; Nippon Data General, Tokyo, Japan, or PC-9801RA; NEC, Tokyo, Japan) for analysis. The cut-off frequency was 10 kHz (3 dB; 24 dB/octave) and the sampling interval for digitizing was 20 µs.
Continuous acquisition of data of unlimited length was possible
with this digitizing speed until the hard disk became full.
Open state probability (Po) and mean open and shut times
were calculated from a continuous record of 3-5 s. Po was measured as the fraction of time in which the current exceeded 50%
of the open amplitude (Barrett et al., 1982). The open and shut
times were defined as the time between a shut-open and the next
open-shut transition and the time between an open-shut and
the next shut-open transition, respectively. The threshold for
shut-open and open-shut transitions was set at 0.5 of the unitary
current amplitude. Mean open and shut times were calculated as
arithmetic averages of the duration of the open and shut times.
To analyze the time course of activation and deactivation, voltage pulses (17 ms in duration) were repeatedly applied to the membrane patch every 1.3 s. Successive records, 100-400 in number, were averaged after leakage and capacitive currents had
been subtracted. Leakage and capacitive currents were measured
from records with no open events or from records in response to
negative voltage steps. To measure the time constants of activation and deactivation, single-exponential curves that fit the rising
or decay phase of the current record were obtained using the
maximum likelihood method with Simplex minimization (Dempster, 1993).
To measure the unitary amplitude, an amplitude histogram
was plotted from a single channel current record of ~320 ms,
and a pair of Gaussian curves that fit the shut and open levels
were obtained using the binned maximum likelihood method
with Simplex minimization (Dempster, 1993). The unitary amplitude was measured from the difference of the means of the two
Gaussian curves.
Distributions of open and shut times were calculated from
three to tens of seconds of continuous records of single-channel currents to analyze gating under activation by Sr2+ or Ca2+. Much
longer records (e.g., extending for more than 100 s and containing 32,000-2,000,000 consecutive open and shut events) were
used to analyze slow blockade. Histograms of open and shut times were plotted with a logarithmic time axis and a square-root ordinate (Sigworth and Sine, 1987). The bin width used was
log(20.125) = log(1.095); i.e., bin No., n, was used to count events
of duration xmin
xmax (in integer sampling periods), 20.125n
xmin, xmax < 20.125(n + 1). Two to several neighboring bins were
combined for very short times of <160 µs; i.e., events with durations of 1, 2, 3, 4, 5, 6, and 7 sampling periods (20 µs) were
counted in combined bins 0-7, 8-11, 12-15, 16-17, 18-19, 20-
21, and 22-23, respectively. Since the durations of the events
were counted as discrete values (i.e., multiples of the digitizing
time) in the computer, the width of each bin (t1 = 20.125(n + 1)
20.125n) of very short duration in the histogram was different from
the actual time range (t2 = xmax
xmin + 1) counted within that
bin. Therefore, the bin count was adjusted by multiplying by t1/t2. These procedures did not affect the results of the analysis because very short events (<0.5 ms) were not used to calculate the
fitting of exponential curves. Histograms in a certain section of
time bins were fitted with exponential curves using the binned
maximum likelihood method (Sigworth and Sine, 1987
; Dempster, 1993
) with Simplex minimization (Dempster, 1993
). Undersampling of short events due to filtering was not corrected because it was not a serious concern for the relative comparisons
shown in Fig. 3 or for the analysis of burst duration shown in Fig.
8. Fitting straight lines were obtained with least-squares methods.
In semilog or log-log plots, logarithm values were used to calculate fitting lines.
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While recording the channel activity for long periods of time,
transient shifts of the gating mode (McManus and Magleby,
1988; Sugihara, 1994
) were occasionally observed. These shifts
were of different types. In one type, very short bursts (<100 µs)
composed of a single or a few open events occurred repetitively.
The shut states that intercalated these very short bursts were either of medium duration (10-500 ms) or were very long (>500
ms). In another type of shift, a reduction of the single-channel
amplitude (to 42%) accompanied the shortening of shut times.
The occurrence of these shifts in the gating mode did not seem
to be affected by increases in [Sr2+]i or the membrane voltage.
The time during which a channel showed a shift in the gating
mode was <10% of the recording time. Periods of transient shifts
in gating were simply omitted for the analysis of slow blockade by
Sr2+.
Solutions
For recordings in the inside-out mode, the pipette (external) solution was composed of 125 mM KCl and 5 mM Hepes (pH adjusted to 7.2 with ~1.5 mM KOH). The bath (internal) solution
was composed of 125 mM KCl and 5 mM Hepes (pH adjusted to
7.2 with ~1.5 mM KOH), plus a given concentration of SrCl2,
CaCl2, or MgCl2 (all from Wako Chemicals, Osaka, Japan). For
Ca2+ concentrations of 2 µM or less, EGTA and CaCl2 were added
to the 125-mM KCl solution in ratios calculated according to Barrett et al. (1982) to give the final desired free concentration. For Ca2+ concentrations of 5 µM or higher, EGTA was not included
in the solution. Ca2+ contamination in nominally Ca-free KCl solution was found to be ~0.5 µM by comparing the open state
probabilities of BK channels in inside-out patches bathed in
nominally Ca2+-free and Ca-EGTA solutions (Sugihara, 1994
).
The BK channels in goldfish hair cells are scarcely activated by
0.5 µM Ca2+, even at +50 mV (Sugihara, 1994
). Contamination
by Ca in SrCl2 is <0.03%. This contamination by Ca2+ was ignored in the analyses of activation because Sr2+ was approximately one-fourth as potent as Ca2+ in activating BK channels
(see RESULTS). Contamination by Ba in SrCl2 is <0.02%. This
equals <2 µM Ba2+ in 10 mM Sr2+ solution. The ability of 2 µM
Ba2+ to produce a slow blocked state is much smaller than that of 10 mM Sr2+, and in fact is nearly negligible. Therefore, Ba2+ contamination was ignored in the analyses of slow blockade produced by Sr2+. When the internal solution in the bath was to be
exchanged, 200 ml of the solution was superfused to completely
wash the content of the chamber (~2 ml).
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RESULTS |
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Activation of BK Channels by Sr2+
Sr2+ activated BK channels in goldfish hair cells in a
voltage- and concentration-dependent manner. The
records of the steady state activities of two BK channels
(conductance, 308 pS) in an inside-out membrane
patch (Fig. 1) show that channels did not open at 20
mV with an internal Sr2+ concentration ([Sr2+]i) of 2 µM, while opening was readily observed at
20 mV
with 10 µM [Sr2+]i. At all of the [Sr2+]i levels, the frequency of opening was markedly enhanced by depolarization of the membrane voltage from
20 (Fig. 1, middle and bottom) to +50 mV (Fig. 1, top). Increased
[Sr2+]i also facilitated opening of the channel at each
voltage.
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Po was measured for a wide range of [Sr2+]i values at
different membrane voltages for the same membrane
patch (Fig. 2 A). Occasional long shut events longer
than 20 ms were omitted from this measurement when
[Sr2+]i was higher than 200 µM and the membrane
voltage was higher than 0 mV since they were likely to
reflect slow blocked states (see below). Po increased
with an increase in [Sr2+]i, as well as with an increase in
the membrane voltage (Fig. 2 A). Po measured under
activation by Ca2+ (at 50 mV) in the same membrane
patch was also plotted (Fig. 2 A, ). The plots for Sr2+
were shifted rightward to a higher concentration than
that for Ca2+ at the same membrane voltage, but the
maximum Po (PoMAX) was the same for Sr2+ and Ca2+
(~0.91 in this case, Fig. 2 A). Fast flickering gating persisted at a saturation level of Sr2+ activation so that
PoMAX never reached 1, as was found for Ca2+ activation
in BK channels of goldfish hair cells (Sugihara, 1994
). The Hill coefficient for activation by Sr2+ was larger for
positive than for negative voltages (1.6 at 50 mV, ~0.7
between
10 and
50 mV; Fig. 2 B). A similar tendency was observed for Ca2+ activation (Sugihara,
1994
). The Hill coefficient for Ca2+ was larger than that
for Sr2+ at the same voltage (2.7 and 1.6 for Ca2+ and
Sr2+, respectively, at 50 mV; Fig. 2 B). This was also reflected in the maximum slope of each concentration-Po
curve, which was less steep for Sr2+ than for Ca2+ at 50 mV (Fig. 2 A).
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When Po was plotted against the membrane voltage for different [Sr2+]i (Fig. 2 C), the resulting S-shaped relations could be roughly fitted with a Boltzmann distribution:
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(1) |
where PoMAX is the saturated value of Po, n is a constant
representing the equivalent number of gating charges,
V0.5 is the membrane voltage at which Po is 1/2 of
PoMAX, V is the applied voltage, and F, R, and T have
their usual meanings. RT/F is 25.4 mV at 22°C. The n
value for the Boltzmann distribution used to fit the
plots for 10-1,000 µM [Sr2+]i ranged between 1.3 and
1.6 (see legend for Fig. 2 C). The n value was slightly
larger for smaller [Sr2+]i as found for [Ca2+]i in the
cloned BK channel (Cui et al., 1997). The plots for Ca2+ obtained from the same membrane patch had a
similar S-shape and were nearly identical to the plots
for Sr2+ at certain higher concentrations; for example,
the plot for 50 µM [Ca2+]i was nearly identical to that
for 200 µM [Sr2+]i, showing that these are equipotent
concentrations. Values of n in six other membrane
patches were between 1.3 and 2.1 and were similar for
Ca2+ (5 µM) and Sr2+ (20-100 µM) in each case. These
results indicate that the voltage dependence of a BK
channel activated by Ca2+ is indistinguishable from that
activated by Sr2+ as long as equipotent concentrations
of Ca2+ and Sr2+ are used to activate the channel to a
similar degree. The n values obtained in the present
channel were similar to those obtained in rat skeletal
muscle (~2.0, Oberhauser et al., 1988
) and in the mslo
BK channel (1.1-1.8, Cui et al., 1997
).
To compare the potencies of Sr2+ and Ca2+ in activating BK channels, V0.5 was measured in eight membrane
patches in voltage-Po plots under several different Sr2+
and Ca2+ concentrations (Fig. 2 D). The slopes of the
concentration-V0.5 plots indicated that a 10-fold increase
in [Sr2+]i and [Ca2+]i produced an ~25- and 35-mV decrease in V0.5, respectively. The concentration-V0.5 plots
for Sr2+ were shifted rightward by approximately fourfold from those for Ca2+ in all cases, indicating that
Sr2+ was approximately one-fourth as potent as Ca2+
(Fig. 2 D). This difference in potency was greater at
higher concentrations of [Sr2+]i and [Ca2+]i due to the
different slopes of the plots (Fig. 2 D, squares). This is
related to the finding that the Hill coefficient for Sr2+ is
smaller than that for Ca2+ (Fig. 2 B). Flattening of concentration-V0.5 plots at high (50-1,000 µM) [Ca2+]i
(Wei et al., 1994; Cui et al., 1997
) or [Sr2+]i was not obvious in the present experiments.
Mean open and shut times were plotted against
[Sr2+]i and membrane voltage in a membrane patch
with a single BK channel (Fig. 2, E and F) to examine
the effects of Sr2+ on the gating of BK channels. The
decrease in the mean shut time was much greater than
the increase in the mean open time when the channel
was activated by increased [Sr2+]i or membrane voltage. This is similar to the changes that occur when the
channel is activated by Ca2+ (Sugihara, 1994).
The distributions of open and shut times were calculated to further examine the difference in the gating of the BK channel activated by Ca2+ and Sr2+. 5 µM [Ca2+]i and 20 µM [Sr2+]i produced similar Po values (0.309 and 0.315 for Sr2+ and Ca2+, respectively, at 50 mV) in the BK channel shown in Fig. 3, indicating that these concentrations of Sr2+ and Ca2+ were equipotent in this channel. The steady state activities recorded with 20 µM Sr2+ and 5 µM Ca2+ at 50 mV were almost the same (Fig. 3 A). Distributions of open and shut times obtained from long records under these conditions are shown in Fig. 3, B and C. While the open-time distributions were rather simple, with a peak at ~0.1 ms (Fig. 3 B), the shut-time distributions had a wide distribution due to some long shut events of 10-600 ms in duration (Fig. 3 C). The histograms for Sr2+ and Ca2+ were nearly identical to each other for both open and shut times, including the above points. Similarly, the open and shut time distributions obtained at different membrane voltages with the same [Sr2+]i and [Ca2+]i resembled each other (Fig. 3, D-G). Similar findings were observed in three other BK channels.
The time courses of activation and deactivation after
voltage steps differ greatly depending on [Ca2+]i in BK
channels (Sugihara, 1994). I examined whether Sr2+
and Ca2+ produce different time courses of activation
and deactivation. Current records from a single BK
channel in response to voltage steps from the holding
voltage of
50 to +50 mV were averaged to investigate
the time courses of activation and deactivation under
the presence of 20 µM Sr2+ or 5 µM Ca2+ (Fig. 4).
These concentrations of Sr2+ and Ca2+ produced
roughly similar activation in the steady state in this BK
channel. There was no clear difference in the responses of channel activity seen in the current traces
for Sr2+ and Ca2+ (Fig. 4, A, a and B, a). The time
courses of the average current for Sr2+ and Ca2+ resembled each other (Fig. 4, A, b and B, b). The activation time constants (1.22 and 1.14 ms) and deactivation
time constants (0.61 and 0.54 ms) for Sr2+ and Ca2+
were close to each other, indicating that the time
courses of activation and deactivation for equipotent
Sr2+ and Ca2+ were nearly identical. Similar results
were obtained in another experiment.
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These results showed that while Sr2+ and Ca2+ differ with regard to potency and the Hill coefficient in activating the channel, equipotent concentrations of these divalent cations produce nearly identical activity in BK channels. Remarkable similarity was demonstrated with regard to PoMAX, voltage dependence of activation, steady state gating kinetics, and the time courses of voltage- dependent activation and deactivation. Note that the above analysis of activation was performed mainly at relatively low [Sr2+]i, and that the production of slow blocked events at high [Sr2+]i was not addressed in this section.
Fast Blockade of BK Channels by Sr2+
Unitary current amplitude of the BK channel is nearly
linearly related to the membrane voltage in the voltage
range between 50 and +100 mV when activated by
low [Ca2+]i (Sugihara, 1994
). This was also true when
the channel was activated by low [Sr2+]i (5-20 µM),
and single-channel conductances under activation by
Ca2+ and Sr2+ were identical. Under higher [Sr2+]i,
however, the current amplitude deviated from the original linear relation in a voltage- and concentration-dependent manner (Fig. 5). In the case shown in Fig. 5,
the original single-channel current amplitude was 13.2 and 26.1 pA at 50 and 100 mV, respectively, with 5 µM
[Ca2+]i. Corresponding values for 0.1, 1.0, and 10 mM
[Sr2+]i were 12.3 and 23.7, 10.9 and 19.7, and 8.0 and
9.8 pA, respectively. Thus, the reduction was greater
with higher [Sr2+]i and at larger membrane voltage.
The amplitudes measured in the same channel at several voltages and [Sr2+]i are plotted in Fig. 6 A. Since
there was no shift in the reversal voltage in the current-
voltage relation in Fig. 6 A, it could be concluded that,
while it produced fast blockade, Sr2+ could scarcely
permeate the channel. Since there was no apparent increase in the noise level in the current during open
events (Fig. 5, G and H), the blockade should be very
fast.
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Ferguson (1991) has shown that Woodhull equation
(Woodhull, 1973
) fits the voltage-dependent reduction
in current amplitude in BK channels in mammalian
cultured skeletal muscle membrane at a given concentration of internal divalent cations (Mg2+, Ca2+, Ni2+,
Sr2+), whereas this equation cannot reflect concentration-dependent reduction without changing the value
of the dissociation constant. In the present study, I first
made a Hill plot to analyze the concentration-dependent reduction in the unitary current amplitude at
each membrane voltage (Fig. 6 B). Data obtained at
concentrations lower than 0.5 mM or at voltages lower
than 30 mV were discarded because scattering became
large due to the very small degree of current reduction.
The slopes of the fitting straight lines (i.e., Hill coefficients) were ~0.7 for all voltages (0.62-0.75, Fig. 6 B).
The reduction in amplitude was then plotted against the voltage (Fig. 6 C). The linear relationship of the plots indicated that Woodhull equation could fit the data at each [Sr2+]i:
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(2) |
where iO and iB are the amplitudes of single-channel
currents in the absence and presence of blocker, respectively, [B] is the concentration of the blocking ion
(Sr2+ in this case), Kd(F)(0) is the dissociation constant
of the ion for fast blockade at 0 mV, z is the effective
charge in valence of the blocking ion, d is the fraction
of the voltage difference at the binding site as measured from the intracellular side of the membrane, and
V is the voltage difference across the membrane (Woodhull, 1973). However, Eq. 2 could not fit the data with
different [Sr2+]i without changing Kd(F)(0) as in skeletal
muscle BK channels (Ferguson, 1991
). Therefore, the
Woodhull equation was modified by incorporating the
Hill coefficient to address the amplitude reduction at
different [Sr2+]i:
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(3) |
where N is the Hill coefficient. The fitting straight lines
in Fig. 6 C had slopes between log(e0.019-fold) and
log(e0.016-fold) mV1 (mean, log(e0.018-fold) mV
1), giving a zd value of 0.45. The Hill coefficient obtained
from the data was 0.74 (average from plots for 100-80
mV). Kd(F)(0) for the best fit was 58 mM. Using these
parameters, the modified Woodhull equation (Eq. 3)
showed a good fit to the amplitude reduction at all of
the [Sr2+]i values tested (curves in Fig. 6 A).
Analyses carried out with high [Sr2+]i in four membrane patches produced basically similar results (Table
I). A Hill coefficient of ~0.7 and a zd value of ~0.5
were obtained in each case. Kd(F)(0) was ~60 mM in
each case except for one. A channel with a conductance of 178 pS had a larger Kd(F)(0) (209 mM) than
the others. This variation in the parameters of fast
blockade among BK channels in the present study may
be related to the nonuniform nature of these BK channels (Sugihara, 1994).
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In two experiments, the effects of Mg2+ and Ca2+ were also measured (Table I, channels three and four). Mg2+ and Ca2+ produced a reduction in the current amplitude very similar to that produced by Sr2+. While the values of N and zd were similar to those for Sr2+ (Table I), the Kd(F)(0) values were slightly different among the divalent cations. The order of the potency of these divalent cations, as represented by the reciprocal of Kd(F)(0), was Mg2+ > Ca2+ > Sr2+. While high [Sr2+]i produced frequent long shut events (see below), Mg2+ and Ca2+ produced such events infrequently. Therefore, 5-10 µM Ba2+ was added to the internal (bath) solution in these experiments to facilitate the measurement of the shut-state current level at depolarized voltages.
Slow Blockade of BK Channels by Sr2+
The duration of shut events decreased significantly
when activation was augmented with an increase in
[Sr2+]i and membrane voltage in BK channels (Fig. 2, E
and F). Thus, shut events longer than 20 ms became
very infrequent when the channel was well activated,
for example, with 20 µM [Sr2+]i at 50 mV (Fig. 3 C).
However, long shut events were observed when the Sr2+
concentration was further increased and a large positive
voltage was applied (Fig. 7). Long shut events became
obvious when the [Sr2+]i concentration was 500 µM or
higher and the voltage was over 30 mV. The activation of
BK channels was nearly saturated at these [Sr2+]i values
and membrane voltages (Fig. 2 A). On the other hand, the frequency of these long shut events increased when
the membrane voltage or [Sr2+]i was increased. These
findings indicate that long shut events are produced by
Sr2+ by a mechanism different from activation. Internal
Ba2+ also produced long shut events in BK channels in
the present study (Fig. 7 D), as has been reported in
other BK channels (Vergara and Latorre, 1983; Miller
et al., 1987
). In the present study, I analyzed the long
shut events produced by Sr2+ in the steady state and examined whether their mechanism was similar to that of
the slow blockade produced by Ba2+. Periods of occasional transient shifts in gating were omitted for these
analyses (see MATERIALS AND METHODS).
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Open- and shut-time histograms were first calculated
from continuous records of single BK channels. Sets of
histograms based on >34,000 open and shut events were
obtained from a membrane patch for different [Sr2+]i
at +30 and +50 mV (Fig. 8). The distributions of open
time (Fig. 8, A-F, row a) were simple and similar in all
cases. Their peak values, which approximately indicated
the mean open time, came at ~1 ms in all cases. On the
other hand, shut-time distributions were not as simple
as open-time distributions. Although most of the shut
events were shorter than 20 ms, shut events longer than 20 ms were seen in all cases. Such long shut events were
infrequent at 500 µM [Sr2+]i and 30 mV (Fig. 8 A, b).
However, with an increase in [Sr2+]i or membrane voltage, these long shut events occurred more frequently, resulting in the formation of a hump in the time range
between 10 and 500 ms in shut-time histograms (Fig. 8,
A-F, row b, arrowheads). This hump was barely seen with
500 µM and 30 mV (Fig. 8 A, b), but became larger with
an increase in [Sr2+]i or voltage (Fig. 8, B-F, row b). It
was impossible to clearly separate long shut events from
ordinary shut events because the hump portion was
continuous with the rest of the histogram. However, the transition seemed to occur at ~10-20 ms. A single-exponential distribution (time constant: l
s), calculated from bins longer than 20 ms, showed a good fit to
the hump portion (i.e., the duration of long shut events)
in each shut-time distribution (Fig. 8, A-F, row b).
Next, I analyzed the `burst' duration since its parameters could provide information for analyzing slow
blockade. To determine burst events, I arbitrarily defined a threshold time for long shut events (4.3-16
ms). This was the time at which the number of long
shut events shorter than this time equaled the number of ordinary shut events longer than this time (Magleby
and Pallotta, 1983b). The numbers of long shut events
and ordinary shut events were given as the area under
or above, respectively, the exponential curve fitting the
long shut events. The accuracy of this threshold time
was not very significant, as will be discussed later. The duration of each burst (i.e., a period bracketed by long
shut events as defined by the threshold) was calculated
from the recorded data to make a histogram of burst
durations (Fig. 8, A-F, row c). Each distribution of burst
durations could be fitted well with a single-exponential
curve (time constant:
b). I assumed that this exponential component accounted for burst events separated by
long shut events. It was obvious that the number of
burst events increased with an increase in [Sr2+]i and
membrane voltage (note the different ordinate scales
in Fig. 8, A-F, row c), in parallel with the increase in the
frequency of long shut events. In addition, the burst
time distribution was shifted to the left with an increase
in [Sr2+]i and membrane voltage. For example, the
peak of the fitting exponentials (i.e., the time constant)
was 1,310 ms for 500 µM [Sr2+]i at 30 mV (Fig. 8 A, c)
and 63.2 ms for 10 mM [Sr2+]i at 50 mV (Fig. 8 F, c).
The burst time distribution did not depend much on
the threshold time for long shut events. For example, in the case of 10 mM Sr2+ and 50 mV, the time constant
of the fitting exponential curve for burst time was 51.0, 63.2, and 94.0 ms for threshold times for long shut
events of 3.32, 6.64, and 15.8 ms, respectively.
With the methods described above, l
s and
b were
measured from current records for different voltages
and different [Sr2+]i in three experiments. These time
constants were plotted against [Sr2+]i in log-log coordinates (Fig. 9 A). These plots were well-fitted with
straight lines (Fig. 9 A). It was obvious that
b was
strongly dependent on [Sr2+]i (slopes of the fitting
lines:
0.74 to
1.23), and was smaller for higher
[Sr2+]i. On the other hand,
l
s did not depend much
on [Sr2+]i (slopes of the fitting lines:
0.15-0.01).
|
Next, I measured the ratio of the occurrence of the
long shut state to the occurrence of other shut events
(n ls/ns). This ratio should be the same as the ratio of
the occurrence of burst events to that of open events,
and is related to the rate constant of the transition
from the nonlong to the long shut state. The area under the exponential curve that fit the long shut events was used as the occurrence of long shut events (n l
s) in
each shut-time histogram and the number of other
shut events (ns) was obtained by subtracting n l
s from
the total number of shut events counted. n l
s/ns was
plotted against [Sr2+]i in log-log coordinates for each
channel in each experiment (Fig. 9 B). These plots
were well-fitted with straight lines, whose slopes ranged
from 0.71 to 1.18.
Interpretation of Slow Blockade Produced by Sr2+
With regard to the mechanism by which long shut
states are produced by high [Sr2+]i, I considered the
single-site model of Vergara and Latorre (1983), which
was originally introduced to explain Ba2+-induced
blockade (Scheme I), where
,
, k1/k
1 [Sr2+]i, and are
rate constants, and k1 and k
1 are dependent on voltage but independent of [Sr2+]i. Since this scheme can
well explain the occurrence of long shut events, these
events will be called slow blocked events (states) in the
following analysis. `Slow' was used to distinguish from fast blockade dealt with in the earlier section. The slow
blocked state is represented by `Blocked,' while `Shut'
and `Open' were meant to represent all of the open
and shut states that occurred during bursts in Scheme
I, although there may have been multiple shut and
open states. Since flickering gating during bursts (see the previous section on activation) was much faster
than the transitions between bursts and slow blocked
events,
and
should be much larger than k1[Sr2+]i
and k
1. Furthermore, flickering gating during bursts was not significantly affected by the changes in [Sr2+]i
or membrane voltages used in this study of slow blockade due to saturation of activation, as indicated by the
following observations. First, distributions of open
time, which was an index for
, were similar for different [Sr2+]i and voltages (Fig. 8, A-F, row a), indicating
that
could be assumed to be unchanged. Second, a
very small change in the probability for the open state
during bursts (P[open|burst]) was observed (0.86 for
500 µM [Sr2+]i at 30 mV and 0.94 for 10 mM [Sr2+]i at
50 mV in the case shown in Fig. 8). Thus,
could also
be assumed to be nearly constant because it was related
to P[open|burst] and
as follows according to Scheme I:
![]() |
(4) |
|
Scheme I predicts that: (a) the duration of the
blocked state and burst time have single-exponential distributions, (b) burst time decreases with increasing Sr2+
concentration, and (c) blocked time is independent of
the Sr2+ concentration (Vergara and Latorre, 1983). All
of these conditions were approximately satisfied by the
experimental results described earlier (Fig. 9, A and B).
To further assess the validity of Scheme I, relations
among [Sr2+]i, b, and n l
s/ns were deduced. In
Scheme I, the burst state stops when the transition
from open to blocked occurs. Thus,
b can be given by
![]() |
(5) |
Since P[open|burst] was nearly constant and k1 is independent of [Sr2+]i, plots of log(b) versus log([Sr2+]i)
should have a slope of ~
1. Meanwhile, n l
s/ns is determined by the rates of transition from open to shut
and open to blocked in Scheme I:
![]() |
(6) |
Since k1 is independent of [Sr2+]i and is nearly constant, plots of log(n1
s/ns) against log [Sr2+]i should
have a slope of ~1. These predictions agreed with experimental measurements (slopes of log([Sr2+]i)
log(
b) plots,
0.74 to
1.23; slopes of log([Sr2+]i)
log(n1
s/ns) plots, 0.71-1.18; Fig. 9, A and B). These results further confirmed the validity of Scheme I for approximating the slow blockade produced by Sr2+.
The voltage dependence of the production of Sr2+ slow blockade was analyzed using the Woodhull equation, which assumes a single binding site within the membrane voltage gradient in the channel pore for blocking and is compatible with Scheme I:
![]() |
(7) |
where Kd(S)(0) is the dissociation constant of the ion for slow blockade at 0 mV, and z and d are as defined in Eq. 2. Based on Scheme I and Eq. 5, Kd(S)(V) is related to the burst duration and slow blocked duration as follows:
![]() |
(8) |
Since P[open|burst] can be considered a constant
(see above), b/
l
s should be proportional to Kd(S)(V) under constant [Sr2+]i. As predicted, plots of log(
b/
l
s)
against membrane voltage at each [Sr2+]i from several
experiments were roughly on straight lines of similar
slope, although there was some scattering (Fig. 9 C). This indicated that the Woodhull equation (Eq. 7) can
account for voltage dependence. In the experiment
shown in Fig. 9 C,
, the mean of the slopes of different [Sr2+]i was log(e0.0381-fold) mV
1, giving a zd value
of 0.95 from Eqs. 7 and 8. By extrapolating the plots to
a voltage of 0 mV, the value of Kd(S)(0) could be calculated. The average Kd(S)(0) value obtained from this experiment was 72 mM, using P[open|burst] = 0.9. The
values of Kd(S)(0) and zd obtained from the two other
experiments plotted in Fig. 9 C were 36 mM and 1.10 (
), and 150 mM and 1.84 (
).
Voltage changes affected b much more than
l
s, like
changes in [Sr2+]i. For example,
b decreased from 425 to 99 ms, while
l
s increased from 29 to 63 ms when
the membrane voltage was increased from 10 to 50 mV
in one case (Fig. 9 C,
). Thus, the voltage dependence of Sr2+-induced slow blockade originated mainly
from changes in k1 in Scheme I, which should be proportional to the reciprocal of
b.
Slow Blocked States Produced by Internal Ba2+
Internal barium ion also produced long shut events in
BK channels in the present study (Fig. 7 D) as has been
reported in other BK channels (Vergara and Latorre,
1983; Benham et al., 1985
; Neyton and Miller, 1988
).
The properties of long shut events produced by Ba2+
were examined in single BK channels that were almost
fully activated by 100 µM [Ca2+]i at positive membrane
voltages. Shut events of long duration occurred frequently with 5 µM [Ba2+]i at 50 mV (Fig. 7 D, note that
the time scale is different from those for other traces).
These long shut events became frequent with an increase in [Ba2+]i and voltage (not shown). Some long
shut events produced by Ba2+ had very long durations
(up to several seconds), while other long shut events
were shorter than 500 ms (Fig. 7 D, arrowheads). The
duration of the latter was similar to that of long shut
events produced by Sr2+.
These long shut events were analyzed by making open- and shut-time histograms from records obtained with different [Ba2+]i (Fig. 10). Shut events longer than 10 ms were rare when no Ba2+ was applied (Fig. 10 A, b). With 5 µM [Ba2+]i, long shut events of 10 ms-10 s were observed (Fig. 10 B, b, arrowheads). With 20 µM [Ba2+]i, the frequency of such long shut events increased, producing an elongated foot in the histogram (Fig. 10 C, b, arrowheads). The component produced by long shut events in the shut-time histogram was distributed over such a wide range of time (roughly 10 ms-10 s), which corresponded to the above observations of very and medially long shut events in the current records, that double exponentials, at least, were necessary to achieve a good fit (Fig. 10). On the other hand, the changes due to differences in [Ba2+]i were small in open-time histograms (Fig. 10, A-C, row a). Similar results were observed with internal Ba2+ in three other BK channels.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three different effects (i.e., activation, fast blockade,
and slow blockade) of internal Sr2+ in BK channels in
goldfish hair cells were examined in the present study.
While only Po was measured in previous studies on Sr2+-induced activation of BK channels (Oberhauser et al.,
1988; Yoshida et al., 1991
), gatings produced by Sr2+
and Ca2+ were compared in the present study. The
findings regarding fast blockade produced by Sr2+ in
the present study were largely similar to the results of a detailed analysis of the fast blockade of skeletal muscle
BK channels produced by Mg2+ and other divalent cations (Ferguson, 1991
). The present study was the first
to analyze in detail the slow blockade produced by Sr2+.
These different effects produced by Sr2+ were apparently independent of each other, indicating that there are specific Sr2+-sensitive sites in the channel molecule related to these effects. The activation, fast blockade, and slow blockade sites may be on the cytoplasmic side of the channel molecule, near the internal entrance of the channel pore slightly within the membrane voltage field, and deep in the channel pore under the strong influence of the membrane voltage field, respectively, as discussed below.
Sr 2+-induced Activation of BK Channels
Sr2+-induced activation of BK channels has been reported in skeletal muscle T-tubule membrane incorporated into planar lipid bilayers (Oberhauser et al.,
1988) and in isolated hippocampal pyramidal cells of
young (7-10 d old) rats (Yoshida et al., 1991
). The dose
at which Sr2+ produced half-maximum activation (Po = 1/2 PoMAX) was 161 and 19.6 times higher than that
needed for Ca2+, respectively, in the former and latter
preparations. In the present BK channel, the concentration of Sr2+ needed to produce half-maximum activation was only approximately four times higher than
that needed for Ca2+. Thus, while the relative potencies
of Sr2+ and Ca2+ for activating BK channels seem to
vary among different BK channels, Sr2+ is always less
potent than Ca2+.
It has been assumed that there are several Ca2+ binding sites in BK channels (Magleby and Pallotta, 1983a;
Moczydlowski and Latorre, 1983
). The difference in
the concentrations of Sr2+ and Ca2+ necessary to activate the channel indicated that the affinity of Sr2+ for
Ca2+ binding sites is about a quarter of that for Ca2+.
However, the dose-response curves for Sr2+ were less
steep than those for Ca2+ (Fig. 2 A). This was also reflected in the difference between the Hill coefficients
for Ca2+ and Sr2+ (Fig. 2 B). A simple explanation for
this finding is that the degree of cooperative interaction of ligand binding sites is weaker for Sr2+ than for
Ca2+, in addition to the difference in affinity. A difference in the slopes of the dose-response curves for Ca2+
and Sr2+ was also observed in skeletal muscle BK channels (Oberhauser et al., 1988
).
A major finding of the present study was the close
similarity of the activities of BK channels evoked by
equipotent concentrations of Ca2+ or Sr2+, despite the
difference in the dose-response curves. Similarity was
observed with regard to maximum Po, the voltage dependence of activation, steady state gating kinetics, and
the time course of pulse-evoked activation and deactivation of average current. This result is in sharp contrast
to the case of the nicotinic acetylcholine receptor cation channel, in which different agonists produce different
gating (Colquhoun and Sakmann, 1985). The present results indicated that the same gating scheme can explain
both Ca2+- and Sr2+-induced activation in BK channels.
If the main or final activation process is not ligand dependent but rather voltage dependent, it would explain why Sr2+ and Ca2+ produced indistinguishable activity in BK channels. The difference in the affinity of
Sr2+ and Ca2+ to the binding sites suggests differences
in the association and dissociation rates of these cations
to the binding sites. The similarity of activity of Sr2+-
and Ca2+-activated channels (Fig. 3) in spite of these
differences may suggest the possibility that each open
or shut state related to each component in open and
shut time histograms does not necessarily correspond
to different numbers of ligands bound, but is mostly determined by intrinsic mechanisms.
The voltage and ligand dependence of the activation
of BK channels have recently been examined in detail
using cloned channels expressed in Xenopus oocytes. It
has shown that the "tail" domain on the carboxyl side
determines apparent sensitivity to the ligand while the
"core" domain on the amino side determines open time, conductance, and, probably, voltage dependence (Wei
et al., 1994). These authors first argued in favor of independent voltage and ligand steps in the activation process on the basis of the flattening in the log([Ca2+]i)
V0.5 relation. A study of macroscopic and single-channel current has shown that Ca2+ and voltage have distinct
effects on activation (DiChiara and Reinhart, 1995
).
Studies of macroscopic and gating current have demonstrated calcium-independent activation (Meera et
al., 1996
) and have suggested the presence of voltage-dependent and ligand-independent steps in the activation process (Toro et al., 1996
; Stefani et al., 1997
). Another study of macroscopic current kinetics indicated the presence of an intrinsic voltage-dependent central
activation process (Cui et al., 1997
). The results of the
present study also support the view that a ligand- and a
voltage-sensitive activation process are partially segregated, and that an intrinsic voltage-dependent activation
process in BK channels is present.
Fast Blockade by Sr 2+
The results of this study show that intracellular Sr2+ reversibly reduces single-channel current through the BK
channel in goldfish hair cells in a voltage- and concentration-dependent manner without changing the zero-current voltage. Ca2+ and Mg2+ also produced similar
current reduction. The potencies of these cations in reducing current amplitude were not very different. A
very similar effect has been observed in cultured rat
skeletal muscle BK channels for Mg2+, Ni2+, Ca2+, and
Sr2+ (Ferguson, 1991). However, in cultured neurons
from rat hypothalamus and brain stem, the potency of
internal Ca2+ was ~1,000× higher than that of internal
Mg2+ in reducing the outward current amplitude (Kang
et al., 1994
). In BK channels from T-tubule membrane
in lipid bilayers, Ca2+ was >10× as potent as Mg2+ or
Sr2+ (Oberhauser et al., 1988
). Therefore, the present
BK channel was similar to the BK channel in cultured
skeletal muscle. While the order of potency was Mg2+ > Ca2+ > Sr2+ in the present material, the order in skeletal muscle was Mg2+ > Ca2+ = Sr2+ (Ferguson, 1991
).
Concerning the speed of blockade, Sr2+ and other divalent cations produced no significant increase in the noise
level, similar to internal tetraethylammonium (TEA) blockade of BK channels (Blatz and Magleby, 1984
;
Yellen, 1984
), while fast blockade of BK channels by internal Na+ is accompanied by an obvious increase in
the noise level in open states (Marty, 1983
; Yellen,
1984
). The original Woodhull equation could account
for the blockade produced by different concentrations of internal TEA (Blatz and Magleby, 1984
), whereas a
modified Woodhull equation was necessary for the fast
blockade produced by internal divalent cations.
It would be reasonable to assume that the site for fast
blockade by divalent cations is near the internal entrance of the channel pore within the membrane voltage field based on the blockade of permeation and the
small zd value. It may be in the vestibule of the channel
pore as proposed by Ferguson (1991). The mechanism of fast blockade by divalent cations is not yet fully understood (Ferguson, 1991
). Voltage-dependent current
reduction at a fixed concentration of divalent cation
could be fitted by the Woodhull equation in the
present study, as in skeletal muscle BK channels (Ferguson, 1991
). Furthermore, the current reduction produced by different Sr2+ concentrations could be fitted
with a modified Woodhull equation with a Hill coefficient value of ~0.7 in the present study. The scheme of
competitive interaction between Mg2+ and the permeation of K+ proposed by Ferguson (1991)
gives a Hill
coefficient of 1, and thus it alone cannot be used to interpret this finding. Although the physical basis for the
modified Woodhull equation (i.e., Hill coefficient of
<1) could not be determined, there are several possible interpretations.
First, a Debye-Hückel ion-ion interaction of electrolytes can be considered. The activity coefficient (f) of
electrolytes decreases with an increase in concentration
([X]), with the relationship log(f) = k[X]1/2, where
the constant k is much larger for divalent ions than for monovalent ions (Bockris and Reddy, 1970
). The k
value for CaCl2 (in M) is 3.2, calculated from known activity coefficient values (f = 0.8588 and 0.7361 for
0.0018 and 0.0095 M of CaCl2, respectively; Bockris and
Reddy, 1970
). If the activity predicted by this relationship, [CaCl2] exp(
3.2[CaCl2]1/2), is used instead of
concentration ([B]) in the original Woodhull equation
(Eq. 2), the predicted Hill plot is slightly convex upward with a tangential slope of <1. Between 1 and 10 mM [CaCl2], the resultant curve could be approximated by the modified Woodhull equation (Eq. 3) with
N = 0.9.
Second, fast blockade by a two-step reaction instead of a single step can be considered. A simple scheme of single-step blockade (Scheme II) fits the Woodhull equation and a Hill coefficient value of 1. However, another model may be possible in which the blocked state is produced by a certain conformational change that can occur after binding of Sr2+ (Scheme III).
|
|
In this scheme, all of the rate constants (k1, k1, k2,
k
2) are large enough for fast blockade, and open* is an open state in which Sr2+ is bound at a site that triggers a conformational change for fast blockade. According to Scheme III, the probability for the channel
to be in the blocked state (PB) is given by:
![]() |
(9) |
where Kd1 = k1/k1 and Kd2 = k
2/k2. The predicted
Hill plot is given by:
![]() |
(10) |
where loge[Sr2+]i and loge([iO iB]/iB) are the coordinates of each data point. Eq. 10 gives a curve convex
upward with a tangential slope of <1. When the Kd1
value is 8 mM, the slope of the curve between 1 and 10 mM [Sr2+]i is ~0.7. Thus, this model can approximate
the experimental results.
Slow Blockade of BK Channels by Sr2+
The kinetics of the slow blockade of BK channels in
goldfish hair cells produced by internal Sr2+ could be
interpreted by a model of a single binding site within the membrane voltage field. The same model has been
used to successfully explain the slow blockade produced by internal Ba2+ in BK channels in various preparations (Vergara and Latorre, 1983; Benham et al.,
1985
; Miller et al., 1987
; Diaz et al., 1996
). The depth of
the Sr2+ binding site within the membrane voltage field
from the internal orifice, as indicated by the zd value in
Eq. 7 divided by 2, was 0.5-0.9 in the present study.
This value is consistent with those reported for Ba2+
blockade (0.8, Vergara and Latorre, 1983
; 0.96, Benham et al., 1985
; 0.5-0.7, Neyton and Miller, 1988
; 0.92, Diaz et al., 1996
). The association rate constant (k1 in
Scheme I) was much more voltage sensitive than the
dissociation rate constant (k
1 in Scheme I) in Sr2+-
induced slow blockade, as observed for Ba2+-induced
blockade, suggesting that the peak of the energy barrier that the blocking divalent cation needs to jump is
quite near the binding site (Vergara and Latorre, 1983
;
Diaz et al., 1996
). Therefore, it would be reasonable to
assume that the site for Sr2+-induced slow blockade lies
within the channel pore and that the energy barrier between the binding site and the internal surface is near
the binding site, as hypothesized for Ba2+-induced
blockade (Vergara and Latorre, 1983
; Neyton and
Miller, 1988
; Diaz et al., 1996
).
Slow blockade in BK channels has also been produced
with internal Ca2+ (Vergara and Latorre, 1983). However, studies with a specific Ba2+ chelator have recently
suggested that this blockade is not caused by Ca2+ but
rather by contamination of the internal solution by Ba2+
(Neyton, 1996
; Diaz et al., 1996
). The slow blockade
produced by Sr2+ in the present study is not likely due
to Ba2+ contamination since (a) the duration of the Sr2+-induced blocked state was different from that of the
blocked state produced by Ba2+, and (b) the amount of
Ba2+ contamination in SrCl2 should be too small to explain the frequent occurrence of slow blocked events under the presence of Sr2+ (see MATERIALS AND METHODS).
In my own analysis of Ba2+-induced blockade in BK
channels of goldfish hair cells, I found that there were
at least two exponential components in the distribution
of the slow blocked events produced by Ba2+. Therefore, I could not apply a simple kinetics analysis to
Ba2+-induced blockade, as was used for Sr2+-induced
slow blockade. Although Ba2+-induced blockade was
not studied further here, the properties of the blockade were roughly similar to those reported in BK channels in skeletal or smooth muscle preparations (Vergara and Latorre, 1983; Benham et al., 1985
; Neyton
and Miller, 1988
; Diaz et al., 1996
), especially regarding
the concentration range of [Ba2+]i (~10 µM), the duration of the blocked state (up to several seconds), and
its dependence on [Ba2+]i and voltage.
The overall similarity of Sr2+- and Ba2+-induced slow
blockade suggests that the binding site for Sr2+ may be
the same as that for Ba2+. Since the Pauling radius of
Sr2+ (1.13 Å) is slightly smaller than those of Ba2+ (1.35 Å) and K+ (1.33 Å), Sr2+ may be able to access the Ba2+
binding site. However, the slow blockade produced by
Ba2+ was more complicated than Sr2+-induced slow
blockade, as indicated by the fact that the durations of
Ba2+-blocked events could not be fitted with single-
exponential curves (Fig. 10). One possible explanation
for this difference is that there may be two Ba2+ binding sites within the channel pore, as has been proposed
in studies on external Ba2+-induced blockade in Shaker
K+ channels (Hurst et al., 1995) and in smooth muscle
BK channels (Sohma et al., 1996
). If a model of multiple Ba2+ binding sites is applicable to Ba2+-induced
blockade in BK channels of the present study, Sr2+ may
bind to one of the multiple Ba2+ binding sites. The differences in the concentration range required to produce slow blockade and in the duration of blocked
events between Sr2+ and Ba2+ could be due to differences in the energy levels of the binding sites and the
barriers to these cations.
![]() |
FOOTNOTES |
---|
Address correspondence to Dr. Izumi Sugihara, Department of Physiology, Tokyo Medical and Dental University, School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. Fax: 81-3-5803-5155; E-mail: isugihara.phy1{at}med.tmd.ac.jp
Received for publication 30 June 1997 and accepted in revised form 21 October 1997.
The author thanks Dr. Taro Furukawa for his advice and encouragement regarding this work.This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
![]() |
Abbreviation used in this paper |
---|
BK channel, large-conductance Ca2+-activated K+ channel.
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