From the Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101-6430
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ABSTRACT |
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Coexpression of the subunit (KV,Ca
) with the
subunit of mammalian large conductance Ca2+-
activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel
analysis to investigate the mechanism for this increase, we found that the
subunit increased open probability
(Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of
the
subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that
the
subunit does not act by increasing all the Ca2+ binding rates proportionally. The
subunit also inhibited
transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the
subunit
that increases the apparent Ca2+ sensitivity of the channel. In the presence of the
subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean
open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to
channels expressed from alpha subunits alone.
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INTRODUCTION |
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Large conductance Ca2+-activated K+ channels (BK
channels)1 play an important role in regulating the excitability of nerve, muscle, and other cells by stabilizing
the cell membrane at negative potentials (reviewed by
Latorre et al., 1989; McManus, 1991
; Kaczorowski et al.,
1996
). BK channels are activated by both intracellular calcium (Ca2+i) and membrane depolarization, and
hence can serve as an important link to couple the effects of Ca2+i and membrane potential, two common
forms of signaling in cells. To facilitate this linkage, the
Ca2+i sensitivity of BK channels, defined as the Ca2+i required for half-maximal activation at a given voltage, is set to match the specific needs of the various cells. In
some tissues, such as smooth muscle, BK channels are
highly Ca2+i sensitive, while in other tissues, such as
skeletal muscle, the channels are much less Ca2+ sensitive (Singer and Walsh, 1987
; McManus and Magleby,
1991
; Tanaka et al., 1997
).
Recent studies have given some insight into the molecular basis for differences in Ca2+ sensitivity. BK channels can be formed of either subunits alone or of
together with
subunits (Adelman et al., 1992
; Garcia-Calvo et al., 1994
; McManus et al., 1995
; Dworetzky et al.,
1996
; Tseng-Crank et al., 1996
). The larger pore-forming
subunits are encoded by the gene at the slo locus,
mutations of which underlie the Drosophila slowpoke
phenotype (Atkinson et al., 1991
; Adelman et al., 1992
;
Butler et al., 1993
; Pallanck and Ganetzky, 1994
; Dworetzky et al., 1994
; Tseng-Crank et al., 1994
; Wallner et al., 1996
). The
(slo) subunit shows homology
with the pore-forming subunits of the voltage-dependent superfamily of K+ channels, which have at least six
putative transmembrane domains, a pore-forming region between S5 and S6, and an S4 voltage-sensor region (Atkinson et al., 1991
; Salkoff et al., 1992
; Butler
et al., 1993
; Jan and Jan, 1997
). However, the NH2- and
COOH-terminal ends of the
subunits differ from
those of the superfamily. The NH2 terminus of mammalian
subunits displays an additional transmembrane domain, S0, that places the amino terminal into
the extracellular space and is required for the action of
the
subunit (Wallner et al., 1996
; Meera et al., 1997
).
The COOH-terminal tail is greatly extended, displays
four hydrophobic domains, and appears to provide the
Ca2+-sensing domain of the channel (Wei et al., 1994
;
Schreiber and Salkoff, 1997
). The
subunit, with two
putative transmembrane domains, shows no homology
with other ion channel subunits (Knaus et al., 1994
).
While subunits assemble as tetramers to form functional channels by themselves (Shen et al., 1994
),
subunits expressed alone do not (McManus et al.,
1995
). Rather,
subunits can associate with
subunits
in a 1:1 stoichiometry (Garcia-Calvo et al., 1994
), increasing the apparent Ca2+ sensitivity of the
subunits
~10-fold (McManus et al., 1995
). It is the presence of
the
subunit that confers the greatly increased Ca2+
sensitivity to BK channels in smooth muscle (McManus
et al., 1995
; Tanaka et al., 1997
). Although it is known
that the
subunit slows activation and deactivation kinetics (Dworetzky et al., 1996
; Meera et al., 1996
;
Tseng-Crank et al., 1996
), while having little effect on
channel open probability in the absence of Ca2+i
(Meera et al., 1996
), the mechanism by which the
subunit increases the apparent Ca2+ sensitivity of BK
channels is not known. The
subunit could increase
apparent Ca2+ sensitivity through fundamental changes
in the gating mechanism, such as by generating additional conformational states or Ca2+-binding sites. Alternatively, the
subunit might act by modulating the
gating of the
subunit to increase the rate of Ca2+
binding or to change the rates of selected transitions
among the various conformational states.
We now use the resolving power of the single-channel recording technique to differentiate among these
possible types of action by studying the kinetics of single BK channels comprised of subunits alone, or of
both
and
subunits. Our data suggest that the
subunit does not act by changing the fundamental gating mechanism, as neither the Hill coefficients for Ca2+
binding nor the numbers of detected kinetic states entered during gating were changed by the
subunit.
The data also suggest that the
subunit had little effect
on the initial Ca2+-binding steps involved in activation
of the channel, as the durations of the gaps (the long
closed intervals) between bursts of activity were little
changed. Instead, the
subunit increased Ca2+ sensitivity through selected modulation of transition rates to retain the channel in the open and closed states that
generate the bursts of activity (bursting states), increasing burst duration 20-100-fold. We also found that the
subunit inhibited transitions to subconductance states,
and that the gating of native BK channels from cultured rat skeletal muscle was similar to the gating of BK
channels expressed from
subunits alone.
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METHODS |
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Heterologous Expression of BK Channels in Human Embryonic Kidney 293 Cells
Human embryonic kidney (HEK) 293 cells were transiently transfected with expression vectors (pcDNA3) encoding the subunit (mslo from mouse, Genbank accession number MMU09383) and
the
subunit (bovine
, Genbank accession number L26101) of
BK channels, kindly provided by Merck Research Laboratories,
and also with an expression vector encoding the green fluorescent protein (GFP, Plasmid pGreen Lantern-1; GIBCO BRL).
Cells were transfected transiently using the Lipofectamine Reagent (Life Technologies, Inc.). The GFP was used to monitor
successfully transfected cells. For transfection, cells at 30-40%
confluency in 30 mm recording Falcon dishes were incubated
with a mixture of the plasmids (total of 1 µg DNA), Lipofectamine Reagent (optimal results at 7 µl), and Opti-MEM I Reduced Serum Medium (GIBCO BRL). The mixture was left on
the cells for 1 h, after which it was replaced with standard tissue culture media: DMEM with 5% fetal bovine serum (GIBCO BRL)
and 1% penicillin-streptomycin solution (Sigma Chemical Co.).
The cells were patch-clamped 2-3 d after transfection.
In the coexpression experiments, a fourfold molar excess of
plasmid encoding the subunit was used to drive coassembly
with the
subunits in the expressed channels (McManus et al.,
1995
). Using the same promoter (cytomegalovirus) for the
and
subunits and the GFP increased the probability that if the GFP
was expressed, the included subunits would also be expressed.
While we did not prove directly that all BK channels studied from
cells cotransfected with plasmids encoding for
and
subunits
were indeed composed of both
and
subunits, the markedly
different bursting kinetics of BK channels from such cells (see
RESULTS) indicated that the coexpression of the
with the
subunit altered the gating of the channels.
Solutions
The intracellular solution contained 175 mM KCl, 5 mM TES
[N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid] pH
buffer, and 10 mM EGTA and 10 mM HEDTA to buffer the Ca2+
(see below). The extracellular solution contained either 150 or
175 mM KCl and 5 mM TES and had no added Ca2+ or Ca2+ buffers. Both the intracellular and extracellular solutions were brought to pH 7. The amount of Ca2+ added to the intracellular
solution to obtain approximate free Ca2+ concentrations of 0.1-
100 µM was calculated using stability constants for EGTA from
Smith and Miller (1985) and for HEDTA from Martell and Smith
(1993)
. These solutions were then calibrated using a Ca2+ electrode (Ionplus from Orion Research, Inc.) standardized against solutions with KCl and TES (as in the experimental solutions) in
which a known amount of Ca2+ was added. Before adding Ca2+,
any contaminating divalent cations were removed from the solution by treatment with Chelex 100 (Bio-Rad Laboratories). The
solutions bathing the intracellular side of the patch were
changed by means of a valve-controlled, gravity-fed perfusion system using a microchamber (Barrett et al., 1982
).
Single-Channel Recording and Analysis
Currents flowing through single BK channels in patches of surface membrane excised from HEK 293 cells transfected with
clones for either or
and
subunits were recorded using the
patch-clamp technique (Hamill et al., 1981
). All recordings were
made using the excised inside-out configuration in which the intracellular surface of the patch was exposed to the bathing solution. BK channels were identified by their large conductance and
characteristic voltage and Ca2+ dependence (Barrett et al.,
1982
). Endogenous BK channels in nontransfected HEK 293 cells were not seen, but we cannot exclude that they might exist
at a low density. Currents were recorded with an Axopatch 200A
amplifier (Axon Instruments) and stored on VCR tapes using a
VR-10B digital data recorder. The currents were then analyzed
using custom programs written in the laboratory. Single-channel
patches were identified by observing openings to only a single
open-channel conductance level during several minutes of recording in which the open probability was >0.4. Except for two
experiments in which patches containing two BK channels were used to measure the effect of Ca2+ on open probability (Po), all
data were from patches containing a single BK channel. Experiments were performed at room temperature (20-25°C).
Single-channel current records were low-pass filtered with a
four-pole Bessel filter to give a final effective filtering of typically
4.5-10 kHz (3 dB) and were sampled by computer at a rate of
125-250 kHz. The methods used to select the level of filtering to
exclude false events that could arise from noise, measure interval durations with half-amplitude threshold analysis, and use stability plots to test for stability and identify activity in different modes
have been described previously, including the precautions taken
to prevent artifacts in the analysis (McManus et al., 1987
; McManus and Magleby, 1988
, 1989
; Magleby, 1992
). The kinetic
analysis in this study was restricted to channel activity in the normal mode, which typically involves ~96% of the detected intervals (McManus and Magleby, 1988
). Activity in modes other than
normal, including the low activity mode (Rothberg et al., 1996
),
was removed before analysis, as were transitions to subconductance levels, except when the subconductance levels were being
studied specifically. The numbers of intervals during normal activity analyzed for each experimental condition ranged from
~1,500 to ~200,000, with the greater numbers of intervals being
obtained for higher Ca2+i where the channel activity was higher.
The methods used to log-bin the intervals into dwell-time distributions, fit the distributions with sums of exponentials using maximum likelihood fitting techniques (intervals less than two dead times were excluded from the fitting), and determine the number of significant exponential components with the likelihood ratio test have been described previously (McManus and
Magleby, 1988, 1991
; Colquhoun and Sigworth, 1995
). Dwell-time distributions are plotted with the Sigworth and Sine (1987)
transformation, which plots the square root of the number of intervals per bin without correcting for the logarithmic increase in
bin width with time. With this transform, the peaks in the plots
fall at the time constants of the major exponential components.
The method of defining a critical gap (closed interval) in order to identify bursts is detailed in Magleby and Pallotta (1983). In brief, the distributions of closed-interval durations were first
fitted with, typically, the sum of five exponential components. The closed intervals from the one to two exponential components with the longest time constants were then defined as gaps
between bursts, as there was typically a difference of one to three
orders of magnitude in the time constants separating the components generating gaps between bursts from those generating
closed intervals within bursts. A critical time was then defined to
separate closed intervals that were gaps between bursts from
those that were gaps within bursts, so that the numbers of misclassified closed intervals would cancel out. The critical time was
found to be relatively insensitive to the numbers of exponentials
used to fit the dwell-time distribution. Burst analysis was performed on data sets from single channels in which Po was typically less than ~0.8, since it became increasingly difficult to define gaps between bursts as the Po approached its maximum
value of ~0.96 during activity in the normal mode.
Native BK Channels from Cultured Rat Skeletal Muscle
The parameters describing bursting kinetics for native BK channels from cultured rat skeletal muscle were obtained by analyzing data from previous experiments by McManus and Magleby
(1991) and Rothberg and Magleby (1998)
, which can be consulted for the experimental details.
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RESULTS |
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The Subunit Alters the Gating of mslo as Revealed by
Single-Channel Kinetics
The patch-clamp technique was used to record currents from single BK channels in patches of membrane
excised from HEK 293 cells after transfection with either the subunit (referred to as
channels) or with
both
and
subunits (referred to as
+
channels).
The effects of the
subunit on the gating are illustrated in Fig. 1, which shows representative single-channel currents recorded with 1.8, 3.6, or 5.4 µM calcium
at the inner membrane surface (Ca2+i). The activity of
both
and
+
channels increased with increasing Ca2+i, and for each Ca2+i, the presence of the
subunit
further increased the activity. These observations are
fully consistent with earlier studies, using mainly currents through multiple channels, that established that
the
subunit increases the open probability (Po) (McManus et al., 1995
; Dworetzky et al., 1996
; Meera et al.,
1996
; Tseng-Crank et al., 1996
).
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The first clues towards the mechanism by which the subunit increases Po are readily apparent in Fig. 1. The
subunit had marked effects on the single-channel gating kinetics: at a fixed Ca2+i, the
subunit greatly increased the durations of the bursts of activity while appearing to have little effect on the durations of the gaps
(closed intervals) between bursts. This characteristic effect of the
subunit on the bursting kinetics of the single-channel current records was consistently observed
in comparisons of data from 19
channels and 10
+
channels. 10 of these channels (5
and 5
+
)
were then analyzed in detail to obtain the results in the
rest of the paper.
The Subunit Increases Po while Having Little Effect on the
Hill Coefficient
As evident in Fig. 1, +
channels are open a greater
fraction of the time at a given Ca2+i than are
channels. To further examine this difference in Ca2+ sensitivity, we plotted Po vs. Ca2+i for
and for
+
channels. Typical results are shown in Fig. 2 A, where the
Ca2+i for a Po of 0.5 (Kd) was 9.2 ± 2.3 µM (mean ± SD) for the
channels, shifting to 2.6 ± 0.52 µM for
the
+
channels (+30 mV). In a series of similar experiments, the Kd was 14.2 ± 7.2 µM (range: 6.9-22.86
µM, n = 5) for a channels and 3.5 ± 1.3 µM (range:
2.2-4.9 µM, n = 5) for
+
channels. Thus, the effect of the
subunit on Po was equivalent to increasing
Ca2+i approximately fourfold.
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The Hill coefficients derived from the Po vs. Ca2+i
plots like those in Fig. 2 A were 4.6 ± 1.8 for channels
and 4.5 ± 1.6 (mean ± SD) for
+
channels (the
slopes were not significantly different: P > 0.9), suggesting that four to five Ca2+ ions (range: 2-6) typically
bound to activate the BK channels in our experiments.
These values are within the ranges of 2-5 typically observed for both native and cloned BK channels (McManus, 1991
; Cox et al., 1997
, and references therein).
The Subunit Increases Mean Open Time and Decreases
Mean Closed Time
To investigate the basis for the subunit-induced increase in Po, we measured the observed durations of
the open and closed intervals for data obtained from
patches containing either a single
or an
+
channel
over a range of Ca2+i. Since determinations of observed
mean open- and closed-interval duration are highly dependent on the time resolution, comparisons between
specific
and
+
channels were made only for data obtained at the same level of filtering. Results are
shown in Fig. 2, B and C, for a representative comparison, where the
subunit increased mean open times
3-7-fold and decreased mean closed times ~10-fold
over the examined ranges of Ca2+i. Similar results were
found for comparisons between four additional
and
four additional
+
channels, each channel from a
different experiment, paired for the same level of filtering, over a range of filtering (4.5-10 KHz).
Thus, the subunit increases Po through a dual effect of increasing observed mean open times and decreasing observed mean closed times. (It will be shown
in a later section that the decrease in mean closed
times with the
subunit results in large part from a decrease in the frequency, rather than the duration, of the longer closed intervals.) At high levels of Ca2+i, and
consequently high Po, the mean durations of the closed intervals were brief, and the
subunit had less of an effect on the durations of these already brief closed intervals. We did not explore the effects of nominally zero
Ca2+i, where the
subunit has been reported to have
little effect on Po (Meera et al., 1996
).
The Subunit Does Not Change the Number of Detected
Kinetic States Entered during Gating
The gating of BK channels has been described by kinetic schemes in which the channel makes transformations among a number of different kinetic states (e.g.,
McManus and Magleby, 1991; Wu et al., 1995
; Cox et al.,
1997
). To examine whether the
subunit changes the
number of kinetic states entered during gating, we fitted sums of exponential components to dwell-time distributions (frequency histograms) of open and closed
interval durations for four single
and three single
+
channels. The numbers of significant exponential
components required to fit the distributions gives an
estimate of the minimum number of states entered
during gating (Colquhoun and Hawkes, 1981
, 1995
).
(Examples of dwell-time distributions will be presented
in a later section.)
Fig. 3 plots the number of significant exponential
components required to describe the open (A) and
closed (B) dwell-time distributions for channels and
+
channels. The estimates are plotted against the
numbers of analyzed intervals, as the ability to detect
exponential components is dependent on the numbers of intervals analyzed (McManus and Magleby, 1988
).
Estimates of the minimal numbers of open states (the
number of significant exponential components) ranged
from two to four for both types of channels, with the
lower estimates of two open states associated with the
smaller data sets. The mean number of detected open
states for
channels (3.0 ± 0.5; mean ± SD) was not
significantly different (P > 0.38, Mann-Whitney test,
from Snedecor and Cochran, 1989
) from the mean
number of detected open states for
+
channels (3.1 ± 0.6). Estimates of the numbers of detected closed states ranged from three to seven for
channels and from
four to seven for
+
channels, with the estimate of
three associated with a small data set. The mean number of detected closed states for
channels (5.4 ± 0.9)
was not significantly different (P > 0.37, Mann-Whitney
test) from the mean number of detected closed states for
+
channels (5.6 ± 1.0).
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While it cannot be ruled out that changes in the
numbers of kinetic states did occur with the subunit
but were not detected due to overlapping time constants and/or small areas of some of the exponential
components, the data in Fig. 3 do indicate that the pronounced effect of the
subunit on channel activity did not arise from an obvious change in the numbers of detected kinetic states entered during gating. This observation, that the
subunit did not change the numbers
of detected kinetic states, and the observation in a previous section that the
subunit did not change the Hill
coefficients, suggests that the
subunit may exert its effects by changing transition rates among states rather
than through fundamental changes in the gating mechanism, such as changes in the numbers of states or in
the number of Ca2+-binding sites.
The Subunit Greatly Increases Burst Duration
As a first step towards determining which transition
rates may be affected, we examined the effect of the subunit on bursting kinetics, since the single-channel
records in Fig. 1 suggest that the
subunit greatly increases the durations of the bursts. A critical gap
(closed interval between bursts of openings) was used
to identify bursts (see METHODS). Over the examined
range of Ca2+i, the
subunit increased mean burst duration 20-100-fold (Fig. 4 A), while having little effect
on the mean durations of the gaps (closed intervals)
between or within bursts (Fig. 4 B).
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This marked increase in mean burst duration by the
subunit was associated with pronounced increases in
both the mean open times (Fig. 2 B) and in the mean
number of openings per burst (Fig. 4 C). Since the addition of each opening to a burst requires an intervening (brief) closed interval, the numbers of closings per
burst (given by one less than the number of openings
in Fig. 4 C) also increased dramatically. The
subunit-
induced lengthening of bursts decreased the fraction
of the total closed intervals that were gaps between
bursts (Fig. 4 D). For example, with 3.6 µM Ca2+i,
21.7% of the observed closed intervals were gaps between bursts for
channels, and this decreased to 1.8%
for
+
channels. Since the gaps between bursts are
the longer closed intervals, this fractional reduction in
the numbers of such intervals by the
subunit contributes greatly towards the increase in Po by the
subunit.
Since estimates of both mean burst duration and the
mean duration of gaps between bursts were relatively
insensitive to the level of filtering, these parameters
were compared directly for five channels and five
+
channels, each obtained from a patch containing a
single channel, in Fig. 4, E and F. The data from the 10 channels support the representative data shown in Fig.
4, A and B, for one channel of each type: the
subunit
greatly increased mean burst duration while having little effect on the duration of gaps between bursts. While
there was considerable variability in estimates of mean
burst duration among channels of the same type, all of
the individual estimates of mean burst duration for
channels when compared with
+
channels were clearly separated at each examined Ca2+i, differing by
at least an order of magnitude (Fig. 4 E). Hence, the
magnitude of the effect of the
subunit on mean burst
duration was greater than the variability among channels of the same type.
Since the mean open time, the mean number of
openings per burst, and the mean duration of the gaps
within bursts were all highly sensitive to differences in
filtering, we only compared estimates of these parameters for channels that were filtered the same. Results
similar to those in Figs. 2, B and C, and 4, A-D, were
found for four such additional detailed comparisons between and
+
channels, paired for the same
level of filtering. In each case, the
subunit increased
Po by prolonging the bursts through increases in both
the numbers of openings per burst and in the mean
open time. Prolonging the bursts also decreased the fraction of shut intervals that were gaps between bursts
by preventing the channel from entering the longer
closed intervals that separate bursts.
Increasing Po with the Subunit Was Not Equivalent to
Increasing Ca2+
Similar to the effects of the subunit on increasing
mean open time, increasing Ca2+i also increases mean
open times for BK channels (Fig. 2 B; McManus and
Magleby, 1991
) and the numbers of openings per burst
(Fig. 4 C; Magleby and Pallotta, 1983
). Thus, a potential mechanism for the action of the
subunit is that it
may increase the rates at which the channel binds the
activating Ca2+ ions. If the addition of the
subunit increases all the Ca2+-binding rates proportionally, then
and
+
channels should display identical kinetics
when the Ca2+i is adjusted to give the same Po for both
types of channels. To examine this possibility, we compared the dwell-time distributions of
and
+
channels at the same Po.
Results are shown in Fig. 5, which presents open
dwell-time distributions on the left and closed dwell-time distributions on the right for both and
+
channels, each at two different Ca2+i. At 1.8 µM Ca2+i,
the Po for the
channel was 0.004 (Fig. 5 A), while the
Po for the
+
channel was 0.15 (Fig. 5 B). The increase in Po induced by the
subunit was due to both a
pronounced shift in the open intervals to longer durations and a marked decrease in the number of longer
closed intervals (gaps between bursts), as indicated by a
decrease in the amplitude of the component marked gaps.
|
By increasing Ca2+i from 1.8 to 5.4 µM, the Po of the
channel was increased from 0.004 to 0.16 (Fig. 5 C)
to approximate the Po of 0.15 for the
+
channel at
1.8 µM Ca2+i (Fig. 5 B). A comparison of the dwell-time
distributions for the
and
+
channels at the same
Po showed marked differences in the kinetics: both the
mean open times and the mean durations of the gaps
between bursts were approximately an order of magnitude less for the
channel (Fig. 5 C) than for the
+
channel (Fig. 5 B), while the relative numbers of gaps
between bursts were greater for the
channel than for
the
+
channel. These marked differences in the kinetics of
and
+
channels at the same Po exclude the possibility that the
subunit acts by the same proportional increases in all the rate constants for Ca2+ binding.
The Subunit Has Little Effect on the Durations of the Gaps
between Bursts
One reason why increasing Po with the subunit was
not equivalent to increasing Ca2+i in the absence of the
subunit was the differential effects of the
subunit
and Ca2+i on the gaps between bursts. Fig. 4, B and D,
shows that the
subunit had little effect on the durations of the gaps between bursts, while decreasing their
relative numbers. This can also be seen in Fig. 5, where
the addition of the
subunit at a fixed Ca2+i had little
effect on the mean durations of the gaps between bursts (positions of the peaks labeled gaps) while it decreased the relative numbers of the gaps, as indicated
by the decrease in amplitude of the peaks in the presence of the
subunit (compare Fig. 5 A to B for 1.8 µM
Ca2+i and Fig. 5 C to D for 5.4 µM Ca2+i).
Ca2+i Decreases the Durations of the Gaps between Bursts
In contrast to the little effect of the subunit on the
durations of the gaps between bursts, increasing Po by
raising Ca2+i decreased the durations of gaps between
bursts for both
and
+
channels. This decrease is
shown in Fig. 4 B, where increasing Ca2+i reduced the
durations of gaps between bursts for both types of
channels. This effect of Ca2+i in reducing the durations
of gaps between bursts for both the
and
+
channels can also be seen in the dwell-time distributions in
Fig. 5, where increasing Ca2+i from 1.8 to 5.4 µM
shifted the peaks labeled gaps to briefer durations
(compare Fig. 5 A to C for
channels and Fig. 5 B to D
for
+
channels). Thus, a major means by which
Ca2+i increases Po for both
and
+
channels is to
drive the channels from the gaps between bursts into
the bursting states, decreasing the durations of the
gaps between bursts.
The Subunit Acts Specifically to Stabilize Bursting Activity
The results in Figs. 1 and 4 A showed that burst duration was markedly greater for +
channels than for
channels for data obtained at the same Ca2+i. The results
also showed that increasing Po by increasing Ca2+i increased burst duration for both
and
+
channels.
Since the
subunit increases Po (Fig. 2 A), the greater
burst duration for
+
channels could have been a
consequence of the increased Po, rather than a specific
effect of the
subunit on lengthening the bursts.
To distinguish between these two possibilities, the effects of the subunit on the bursting kinetics were
studied at the same Po for
and
+
channels over a
wide range of Po, obtained by changing Ca2+i. The results are shown in Fig. 6, where the parameters describing bursting kinetics are plotted against Po. When
and
+
channels were compared at the same Po (the
Ca2+i was higher for the
channels to obtain the same
Po), mean burst duration was still greatly increased for
+
channels when compared with
channels (Fig. 6
A), due mainly to increases in both mean open times
(Fig. 6 C) and the mean number of openings per burst (Fig. 6 D). Thus, the
subunit directly facilitates bursting, as its effects on bursts are greater than if the Po
were elevated to the same level in the absence of the
subunit by increasing Ca2+i.
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In contrast to the subunit-induced increases in the
burst parameters, the mean durations of the gaps
(closed intervals) between bursts were about an order
of magnitude less for
channels than for
+
channels at the same Po (Fig. 6 B). Since the
subunit has
little effect on the durations of the gaps between bursts
(Figs. 4 B and 5), this difference reflects the fact that the
data from
channels were obtained at a higher Ca2+i
to obtain the same Po. The higher Ca2+i for the
channels reduced the durations of the gaps between bursts.
Consistency of Bursting Kinetics as a Function of Po
Fig. 7, A and B, plots the mean burst duration and the
mean duration of the gaps between bursts against Po
for five and five
+
channels. Po ranged from
~0.0003 to ~0.85. The points cluster around the lines
(linear least squares fits to the log of the points), indicating a relative lack of variability when these bursting
parameters are plotted against Po. This can be compared with the data in Fig. 4, E and F, where the variability is greater when the same bursting parameters
are plotted against Ca2+i. Nevertheless, for both types
of plots, the variability among channels of the same
type was less than the effect of the
subunit on the indicated bursting parameters.
|
A variability in the Ca2+ dependence of BK channels
has been described previously for native, purified, and
cloned channels (Moczydlowski and Latorre, 1983;
Singer and Walsh, 1987
; McManus and Magleby, 1991
;
Giangiacomo et al., 1995
; Silberberg et al., 1997). Plotting the bursting parameters against Po rather than Ca2+i
may provide a means to remove much of this variability
when studying the detailed single-channel kinetics.
Native BK Channels from Cultured Rat Skeletal Muscle Have
Bursting Kinetics Like Channels
The significant separation of the bursting parameters
between and
+
channels, together with the relative lack of variability in the parameters for channels of
the same type (Fig. 7, A and B), makes it possible to
functionally identify whether native BK channels are
composed of
subunits alone, of both
and
subunits, or of a mixture of the two. Bursting parameters
for data from six patches from cultured rat skeletal
muscle, each containing a single BK channel, are plotted in Fig. 7, C and D. The dotted lines replot the continuous lines from Fig. 7, A and B, defining the bursting parameters for the cloned
and
+
channels.
The symbols for the native channels are in the immediate vicinity of the line for the bursting parameters of
channels. The simplest explanation of these observations is that native BK channels from cultured rat skeletal muscle are composed of
subunits alone. This conclusion is consistent with the studies of Tseng-Crank et al.
(1996)
and Chang et al. (1997)
who found low or no
mRNA expression in human, canine, or rat skeletal
muscle. We cannot exclude, however, that the native
channels may have one or more
subunits per channel, but appear to gate like
channels because of other
factors. For example, the alternative splice structure of
BK channels can alter gating (Lagrutta et al., 1994
).
Since the structure of the studied native BK channels is
not known, they may be of a different splice variant
than the cloned channels.
The Subunit Inhibits Entry into Subconductance States
Ion channels can enter subconductance levels during
gating, reflecting the entry of the channel into conformations that are not fully open or are perhaps partially
blocked (Barrett et al., 1982; Chapman et al., 1997
;
Premkumar et al., 1997
; Zheng and Sigworth, 1997
).
Fig. 8 shows a typical example of gating to a subconductance level that was observed in
channels, but was seldom observed in
+
channels. To examine the subconductance gating, 210 min of current records from
24
channels and 230 min from 15
+
channels
were visually inspected for transitions to subconductance levels with durations longer than 50 ms. There were 382 transitions to such subconductance levels with
a mean duration of 0.4 s for the
channels, 9 transitions with a mean duration of 0.3 s, and 1 transition
with a duration of 9 s for the
+
channels. The total
time spent in subconductance levels for each channel
was divided by the total time of the recording for that
channel and converted to percentage for the plots in Fig. 8 C.
|
The mean percentage of time that channels spent
gating to subconductance levels (1.1 ± 1.2%, mean ± SD) was decreased 32-fold (P < 0.0003, Mann-Whitney
test) in the
+
channels (0.034 ± 0.097%). While 20 of 24
channels spent >0.1% of their time gating to
subconductance levels, only 1 of 15
+
channels did.
Thus, the
subunit inhibits entry into partially conducting states that give subconductance levels of the
type shown in Fig. 8.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The accessory subunit of mammalian BK channels
greatly increases Ca2+ sensitivity (McManus et al., 1995
;
Dworetzky et al., 1996
; Meera et al., 1996
; Tseng-Crank,
et al., 1996). The single-channel analysis in our study
provides insight into the mechanism for this apparent increase in Ca2+ sensitivity. We found that the
subunit increased burst duration 20-100-fold by increasing
both the number of openings per burst and the mean
open times, while having little effect on the mean durations of the gaps (closed intervals) between bursts.
The bursting kinetics of channels can be described
by the highly simplified Scheme I, where k1 is the rate
constant for entering bursts and k1 is the rate constant
for leaving bursts. For simple models that describe the
basic single-channel properties of the gating of BK
channels, the gaps between bursts in Scheme I are generated by potential transitions among three to eight closed states, and the bursts in Scheme I are generated
by potential transitions among three to four open states
and three to six brief closed states (Magleby and Pallotta, 1983
; McManus and Magleby, 1991
; Wu et al.,
1995
; Rothberg and Magleby, 1998
). Because the data
in our study were recorded from patches containing a
single BK channel, each gap between bursts represents
the sum of the dwell times in the closed states entered
between bursts for that single channel. Some of the
closed states contributing to the gaps between bursts
would be expected to bind Ca2+, with the binding driving the channel through one or more closed states towards the first open state that terminates the gap between bursts. It is this Ca2+ dependence of the closed
states entered between bursts that produces long gaps
between bursts at low Ca2+ and brief gaps at high Ca2+.
Inspection of Scheme I suggests that the subunit
could promote bursting by facilitating the entry of the
channel into bursts (by increasing k1) or by preventing
the channel from leaving the bursts once entered (by
decreasing k
1), or by both actions. If the
subunit acts
to facilitate entry into bursts, then the durations of the
gaps (closed intervals) between bursts should be decreased by the
subunit. Alternatively, if the
subunit acts by retaining the channel in bursts once entered,
then the
subunit should have little effect on the durations of the gaps between bursts, but should decrease
their relative numbers since the channel would enter
the gaps between bursts less often.
|
Our observations that the subunit increased burst
duration and decreased the numbers of gaps between
bursts (Figs. 1, 4 A, and 5) while having little effect on
the durations of the gaps between bursts (Figs. 4 B and
5) suggest that the
subunit had little effect on k1.
Thus, the
subunit increases Po mainly by slowing k
1
to retain the channel in the bursting states.
In contrast to the relative lack of effect of the subunit on the durations of the gaps between bursts, increasing Ca2+i decreased the durations of gaps between
bursts for both
and
+
channels (Figs. 1, 4 B, and
5). Thus, a major means by which Ca2+i increased Po
for both
and
+
channels was to increase k1 to
drive the channels from the gaps between bursts into
the bursting states. Increasing Ca2+i also increased the
durations of the bursts, but not as much as the increase
induced by the
subunit for the same increase in Po
(Figs. 1, 4, and 6).
How might the subunit effectively slow k
1 to retain
the channel in the bursting states? One possibility
would be for the
subunit to add additional states that
are entered during the bursting. Gating in these additional states could then retain the channel in the
bursts. Another possibility would be for the
subunit
to add additional Ca2+ binding sites that would act to
retain the channel in bursts. Our observations that the
subunit did not change the numbers of exponential
components in the dwell-time distributions (Fig. 3) or
the Hill coefficients (Fig. 2) suggest that the
subunit does not act by changing either the numbers of kinetic
states entered during gating or the effective number of
Ca2+ binding sites. (Our observations that the number
of subunits per channel could be doubled, from four
for
channels to eight for
+
channels, without
changing the numbers of detected kinetic states, indicate that the number of states entered during gating is
not necessarily related to the total number of subunits
comprising the
channel.)
The above findings, when coupled with previous observations that the subunit does not appear to change
the effective gating charge (McManus et al., 1995
;
Meera et al., 1996
), suggest that the
subunit acts not
by fundamental changes in the gating mechanism, such as alterations in either the number of Ca2+-binding sites or the number of major conformational
changes, but rather through modulation of the gating
of the
subunits.
One possible way the subunit might modulate the
gating of the
subunits would be through changes in
the Ca2+ binding rates. If the
subunit increased all
the Ca2+-binding rates to the
subunits proportionally,
then increasing Ca2+i sufficiently to obtain the same Po
for
channels as for
+
channels should give the
same single-channel kinetics for both types of channels.
This was found not to be the case, as the durations of
the bursts, the mean open times, the mean numbers of
openings per burst, and the durations of the gaps between bursts were all considerably less for
channels
than for
+
channels at the same Po (Figs. 5 and 6).
Since the subunit does not increase all of the Ca2+-binding rates proportionally, could it act by increasing
a subset of the Ca2+-binding rates? Our observation
that the
subunit had little effect on the durations of
the gaps between bursts suggests that the
subunit has
little effect on the Ca2+-binding rates to the closed
states that dominate the gaps between bursts. This observation does not exclude the possibility that the
subunit may increase some of the Ca2+-binding rates in
the bursting states, but such an effect would require a
differential effect of the
subunit on the bindings of
successive Ca2+.
If the subunit does act by retaining the channel in
the bursting states, then this is functionally equivalent
to imposing a barrier to prevent the channel from leaving the bursting states. If this is the case, then the deactivation from the bursting states that occurs in the presence of Ca2+ after a step to negative membrane potentials might be expected to be slowed by the
subunit.
Consistent with this possibility, the
subunit does slow
deactivation after steps to negative membrane potentials (Dworetzky et al., 1996
; Meera et al., 1996
: Tseng-Crank et al., 1996
).
The subunit of the BK channel bears no sequence
homology with accessory subunits from other channels
(Knaus et al., 1994
), suggesting that modulatory subunits may have evolved separately as needed to modulate specific channels. It also appears that the
subunit
of the BK channel works differently from the modulatory subunits of other channels that increase expression levels and speed activation and inactivation rates
(Lacerda et al., 1991
; Varadi et al., 1991
; Isom et al.,
1992
; Rettig et al., 1994
; Makita et al., 1994
; Heinemann et al., 1995
; Morales et al., 1995
; Shi et al., 1996
).
However, the actions of the
subunit for the BK channel seem to have some features in common with the actions of the accessory Ca2+ channel
2A subunit on Ca2+
channels in the presence of a dihydropyridine derivative; both subunits increase burst duration, although in
the case of the Ca2+ channel this increase occurs only
when the Ca2+ channel is in a high Po mode. Interestingly, these increases in burst duration by the different
subunits on different channels occur even though the
subunit of BK channels has two putative transmembrane
segments (Knaus et al., 1994
), while the
2A subunit of
Ca2+ channels is cytoplasmic (Takahashi et al., 1987
).
A comparison of the bursting kinetics of BK channels
from cultured rat skeletal muscle to the bursting kinetics of channels and
+
channels indicated that BK
channels in cultured rat skeletal muscle have bursting
kinetics similar to
channels (Fig. 7). Thus, BK channels in cultured rat skeletal muscle gate as if they are
composed of
subunits alone. This conclusion is consistent with the studies of Tseng-Crank et al. (1996)
and
Chang et al. (1997)
, who found low or no
mRNA expression in human, canine, and rat skeletal muscle. In
contrast to skeletal muscle, BK channels in tracheal
smooth muscle are composed of
+
subunits, and most BK channels in human coronary artery smooth
muscle function as if they are composed of
+
subunits (Tanaka et al., 1997
). The
subunit would confer
a greater Ca2+ sensitivity to BK channels in smooth muscle.
Our observation that the subunit of BK channels decreases the percentage of time spent in gating to subconductance levels (Fig. 8) suggests that the
subunit of BK
channels stabilizes the full conductance level of the
open states. Similar to our observation for BK channels,
the presence of an auxiliary subunit for the ryanodine
receptor also decreases the percentage of time spent in
gating to subconductance levels (Ondrias et al., 1996
).
Conclusion
From a functional viewpoint, it is the retention of the
BK channel in the bursting states by the subunit that
increases the apparent Ca2+ sensitivity of the channel.
In the presence of the
subunit, each burst of openings
is greatly amplified in duration through increases in both
the numbers of openings per burst and in the mean
open times. The physical mechanism by which the
subunit retains the channel in the bursting states is not
known, but one possibility is that selective allosteric effects of the
subunits on the
subunits facilitate some
conformational changes and/or inhibit others. This selective facilitation and/or inhibition would work to increase the effective energy barrier for leaving the bursting states, through increases in both mean open time and the numbers of openings per bursts.
![]() |
FOOTNOTES |
---|
Address correspondence to Karl L. Magleby, Ph.D., Department of Physiology and Biophysics, R430, P.O. Box 016430, Miami, FL 33101-6430. Fax: 305-243-6898; E-mail: kmagleby{at}mednet.med.miami.edu
Original version received 15 October 1998 and accepted version received 4 December 1998.
We thank Merck Research Laboratories for providing the mslo (initially cloned by Pallanck and Ganetzky, 1994) and bovine
clones used for transfection, and S. Sine for providing helpful advice on the HEK 293 cell expression system.
This work was supported in part by grants from the American Heart Association, Florida Affiliate (C.M. Nimigean), the National Institutes of Health (AR32805 to K.L. Magleby), and the Muscular Dystrophy Association.
![]() |
Abbreviations used in this paper |
---|
BK channel, large conductance Ca2+-activated K+ channel; GFP, green fluorescent protein; HEK cells, human embryonic kidney cells.
![]() |
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