Previous Article | Next Article 
Volume 16, Number 14,
Issue of July 15, 1996
pp. 4344-4359
Copyright ©1996 Society for Neuroscience
[Ca2+]i Elevations Detected by BK
Channels during Ca2+ Influx and Muscarine-Mediated Release
of Ca2+ from Intracellular Stores in Rat Chromaffin
Cells
Murali Prakriya,
Christopher R. Solaro, and
Christopher J. Lingle
Washington University School of Medicine, Department of
Anesthesiology, St. Louis, Missouri 63110
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Submembrane [Ca2+]i
changes were examined in rat chromaffin cells by monitoring the
activity of an endogenous Ca2+-dependent protein:
the large conductance Ca2+- and voltage-activated
K+ channel (also known as the BK channel). The
Ca2+ and voltage dependence of BK current
inactivation and conductance were calibrated first by using defined
[Ca2+]i salines. This
information was used to examine submembrane
[Ca2+]i elevations
arising out of Ca2+ influx and muscarine-mediated
release of Ca2+ from intracellular stores. During
Ca2+ influx, some BK channels are exposed to
[Ca2+]i of at least 60 µM. However, the distribution of this
[Ca2+]i elevation is
highly nonuniform so that the average
[Ca2+]i detected when all
BK channels are activated is only ~10 µM.
Intracellular dialysis with 1 mM or higher EGTA
spares only the BK channels activated by the highest
[Ca2+]i during influx,
whereas dialysis with 1 mM or higher BAPTA blocks
activation of all BK channels. Submembrane
[Ca2+]i elevations fall
rapidly after termination of short (5 msec) Ca2+
influx steps but persist above 1 µM for several
hundred milliseconds after termination of long (200 msec) influx steps.
In contrast to influx, the submembrane
[Ca2+]i elevations
produced by release of intracellular Ca2+ by
muscarinic actetylcholine receptor (mAChR) activation are much more
uniform and reach peak levels of 3-5 µM. Our
results suggest that during normal action potential activity only
10-20% of BK channels in each chromaffin cell see sufficient
[Ca2+]i to be
activated.
Key words:
BK channels;
calcium;
calcium channels;
calcium
stores;
chromaffin cells;
catecholamine secretion;
K+
channel inactivation
INTRODUCTION
The free cytosolic concentration of
Ca2+
([Ca2+]i) is an important
regulator of numerous cellular functions, including neurotransmitter
release, activation of ion channels, and cell death. Because elevations
of [Ca2+]i have such a
wide range of possible consequences, it is to be expected that cells
would have developed methods to trigger the various
Ca2+-dependent events independently of one
another. However, exactly how this is accomplished is not entirely
clear. What is known is that the spread of free
Ca2+ away from Ca2+ sources
is limited to short distances (Allbritton et al., 1992
) and that the
Ca2+ sensitivities of intracellular
Ca2+-binding target proteins vary widely (Kasai,
1993). These observations have led to suggestions that localized
Ca2+ elevations in the immediate vicinity of the
various Ca2+-dependent proteins must be of
central importance (Chad and Eckert, 1984; Simon and Llinas, 1985;
Augustine and Neher, 1992a). If so, appreciation of the specific
consequences of Ca2+ elevations requires the
elucidation of both the features of the
[Ca2+]i elevation, i.e.,
the magnitude, time course, and the spatial spread of
Ca2+, and a knowledge of the precise localization
and the Ca2+ sensitivity of the
Ca2+-dependent target proteins. The present study
examines the relationship between elevations of the submembrane
[Ca2+]i in chromaffin
cells and one such Ca2+-dependent target protein,
the BK channel.
Amplitudes of [Ca2+]i
elevations traditionally have been estimated using exogenous
fluorescent Ca2+ buffers (Grynkiewicz et al.,
1985). Although these buffers can monitor changes in bulk
[Ca2+]i that follow
physiological stimuli, limitations in the methods used to detect
fluorescent signals make it difficult to follow the localized
Ca2+ gradients predicted to occur near individual
Ca2+ channels. Furthermore, introduction of these
exogenous buffers alters many features of the
[Ca2+]i elevations (Sala
and Hernandez-Cruz, 1990; Zhou and Neher, 1993). Therefore, there is
increasing interest in using intrinsic
Ca2+-dependent proteins or processes to define
the amplitudes and time course of
[Ca2+]i elevations. In
the squid giant axon (Adler et al., 1991) and frog hair cells (Roberts
et al., 1990), this approach has supported the theoretical expectation
that [Ca2+]i elevations
during Ca2+ influx are large and rapid.
Furthermore, these studies have suggested that particular
Ca2+-dependent processes are coupled closely to
sites of Ca2+ influx.
BK channels sense rapid changes in both membrane voltage and
submembrane [Ca2+]i and
contribute to action-potential termination in a number of cell types
(Lancaster and Adams, 1986; Lang and Ritchie, 1987), including rat
chromaffin cells (Solaro et al., 1995). In the present study, BK
channels were used to examine submembrane
[Ca2+]i elevations in rat
chromaffin cells. The Ca2+ sensitivity of two
features of BK current, the rate of inactivation and the fractional
activation, was defined by direct introduction of defined
[Ca2+]i into cells. Then
this information was used to determine the amplitude, spatial
distribution, and time course of submembrane
[Ca2+]i elevations during
depolarization-induced Ca2+ influx and
muscarine-mediated elevation of
[Ca2+]i. Our results
indicate that, whereas some BK channels are located close to
Ca2+ channels and are activated rapidly by the
high [Ca2+]i that occurs
around open Ca2+ channels during influx, most BK
channels are located at a distance and are activated slowly by the bulk
[Ca2+]i.
MATERIALS AND METHODS
Chromaffin cell culture. Methods of rat chromaffin
cell isolation and maintenance of chromaffin cell cultures were as
described in earlier reports (Neely and Lingle, 1992a; Herrington et
al., 1995; Solaro et al., 1995). These were based on procedures
described in several earlier studies (Fenwick et al., 1978; Role and
Perlman, 1980; Livet, 1984).
Electrophysiological methods. Whole-cell and
perforated-patch recordings were performed on cells 2-14 d after
plating the cells, as previously described (Neely and Lingle, 1992a,
Herrington et al., 1995; Solaro et al., 1995). In experiments in which
an intracellular solution of defined
[Ca2+]i was used, the
standard whole-cell recording procedure was used (Hamill et al., 1981).
Whole-cell voltage clamp was controlled with the Clampex program in the
pClamp software package (Axon Instruments, Foster City, CA). Analysis
of whole-cell current traces was done with our own software. Fitting of
current waveforms or extracted data was done using a
Levenberg-Marquardt algorithm for minimization of residuals after
adjustment of function parameters.
In perforated patch-clamped experiments, uncompensated series
resistances (Rs) were typically in the
range of 8-15 M
, of which 80-90% was electronically compensated.
Values for uncompensated series resistance and percentage of
compensation are provided in the figure legends. Given the large size
of the BK currents in most chromaffin cells, even 2 M of
uncompensated Rs can result in appreciable
voltage errors. Therefore, analysis was limited to those cells in which
voltage errors resulting from the residual uncompensated
Rs were <20 mV. Series resistances with
the standard whole-cell method were in the range of 4-8 M, of which
80% was compensated.
Solutions. The standard extracellular solution
contained (in mM): 140 NaCl; 5.4 KCl; 10 HEPES;
1.8 CaCl2, and 2.0 MgCl2
titrated to pH 7.4 with N-methylglucamine (NMG). In
experiments in which salines with defined
[Ca2+]i were introduced
into cells, CaCl2 was excluded in the external
saline (0 [Ca2+]o). For
perforated patch-clamp experiments, the pipette saline contained the
following (in mM): 120 K-aspartate, 30 KCl, 10 HEPES(H+), and 2 MgCl2
adjusted to pH 7.4 with NMG. Membrane permeabilization was accomplished
with a mixture of amphotericin B (Rae et al., 1991) and pluronic acid,
as described previously (Herrington et al., 1995). Osmolarity was
measured by dew point (Wescor Osmometer, Wescor, Logan, UT) and
adjusted between 290-310. Most experiments were done in the presence
of 100 or 200 nM apamin to eliminate SK currents
from the data traces. In experiments in which a defined
[Ca2+]i was introduced
into the cell, the pipette saline contained the following (in
mM): 140 KCl, 20 KOH, 10 HEPES(H+), and HEDTA or EGTA with added
CaCl2 to make the appropriate free
Ca2+. 10 HEDTA was used for the 20 and 60 µM
[Ca2+]i salines, 5 HEDTA
for the 10 µM
[Ca2+]i saline, and 5 EGTA for the 1 and 4 µM
[Ca2+]i salines. In
experiments in which cells were stepped to +111 mV after a step to +81
mV, the extracellular Na+ was replaced completely
with NMG to prevent Na+ block of BK channels
(Yellen, 1984) that results from intracellular accumulation of
Na+. In the absence of NMG, steps from +81 mV to
+111 mV often produce anomalous inward currents presumably reflecting
the voltage-dependent block of BK channels by intracellular
Na+. Removal of extracellular
Na+ abolishes this effect. Extracellular solution
changes and drug applications were accomplished via a multibarrel
perfusion system, as described previously (Herrington et al., 1995).
Voltages for perforated-patch whole-cell recordings have been corrected
for a +9 mV liquid junction potential resulting from the use of
aspartate-based pipette salines.
Estimates of the maximal available BK current. A
modification of the Hodgkin-Huxley (H-H; 1952) model was used to
estimate the maximal activatable current at +81 mV after any
Ca2+ influx step or conditioning step. After a
prepulse, peak current resulting from a step to +81 mV arises from two
populations of channels: those that already are open at the time of the
step and those that open after the step. Current through the first
population simply decays in accordance with BKi
inactivation properties. Thus,
|
(1)
|
Current resulting from the second population follows the usual
H-H formalism and can be described by:
|
(2)
|
in which 
h in both equations 1 and 2 represents the inactivation time constant of the channels,
m is the activation time constant,
n is the cooperativity in activation,
Iinst represents the instantaneous current
amplitude, and Imax, the total activatable
BK current in the cell. Together, these equations contain one
additional free parameter, i.e., Iinst,
over the usual H-H formalism, but this parameter is well defined by
the current at t = 0. BK currents activated after steps
to +81 mV were fit, therefore, with the following:
|
(3)
|
Finally, an additional term was included to account for the
small amount of steady-state voltage-dependent, but
Ca2+-independent current active after
inactivation of BK current.
RESULTS
Characteristics of Ca2+-dependent K+
currents in rat chromaffin cells
Rat chromaffin cells display two major categories of
Ca2+-dependent K+ current:
one current results from voltage-dependent, large conductance BK
channels (Neely and Lingle, 1992a), and the other results from
voltage-independent, small conductance SK channels that are sensitive
to apamin (Neely and Lingle, 1992a; Park, 1994). Activation of both
types of current is observed with depolarizing steps that produce
Ca2+ influx via voltage-dependent
Ca2+ channels (Neely and Lingle, 1992a) and with
the release of intracellular calcium by agents such as muscarine and
caffeine (Neely and Lingle, 1992b; Herrington et al., 1995). Over the
range of physiological potentials (
60 to +50 mV), chromaffin cell SK
channels are more sensitive to Ca2+ than BK
channels: maximal activation of SK channels occurs at
[Ca2+]i of 2-4
µM (Park, 1994), whereas maximal activation of
BK channels at 4 µM
[Ca2+]i occurs only at
membrane potentials of +90 mV and above (see Fig. 3). Thus, BK channels
may be more suitable for monitoring submembrane
[Ca2+]i elevations that
exceed 4 µM. In the present study, we have used
BK current to study submembrane
[Ca2+]i elevations; SK
current was blocked with 100 nM apamin.
Fig. 3.
Estimation of instantaneous and peak currents.
A, Cells were stepped for 20 msec to 9 mV and then stepped
to +81 mV for 200 msec on the left and 50 msec on the
right. A cell was clamped with the perforated-patch method
in a, whereas in b the cell was clamped with the
standard whole-cell method with a pipette containing 10 µM
[Ca2+]i. On the
left, currents were digitized with a 500 µsec sampling
interval and, on the right, with a 50 µsec sampling
interval. In both cases, the step to 9 mV results in activation of
outward current. However, with more rapid sampling, the step to +81 mV
first reveals more rapid changes in current reflecting the finite
voltage settling time and, subsequently, the more slowly developing
current reflecting BK current activation. In a, as shown on
the right, ~300 µsec is required for the complete
development of the ``instantaneous'' current. Because of the slower
voltage-settling time of cells clamped with the perforated-patch
method, current at 500 µsec provides a reasonable estimate of the
ohmic component of BK current after the step to +81 mV. Open
arrow in each case indicates the measured instantaneous
current. In b, by contrast, only ~100 µsec is required
for the full development of the ``instantaneous current.'' For
clarity, only the first few points after the voltage step to +81 mV are
shown, and the complete current traces are shown in the insets.
An open arrow in each case indicates the measured
instantaneous current. The filled arrow on the
right indicates the 500 µsec point. Sufficient BK current
activation occurs between 100 and 500 µsec so that current at the 500 µsec point will overestimate the instantaneous current. As described
in Materials and Methods, the change in current between 500 µsec and
1 msec was used to calculate the predicted current at 250 µsec,
assuming a linear rate of current activation. This calculated value
(filled arrow on left) provides a
reasonable estimate of the instantaneous current observed with the
faster sampling rate on the right. a,
Rs, 12 M;
Cm, 5 pF; 80% compensated. b,
Rs, 4 M; Cm,
5 pF; 80% compensated. B, BK current activated at +90 mV by
10 µM
[Ca2+]i included in the
pipette was fit by a modified Hodgkin-Huxley model of current
activation-inactivation. This model accounts for BK inactivation that
might occur in the rising phase of the current. The peak current at +90
mV arises from a population of channels already open (the instantaneous
current component), which decays in accordance with
BKi inactivation properties (indicated by the
thin line), and a second population, which follows the
usual H-H formalism (indicated by the thick line).
The true peak current is the sum of the peak current predicted by the
usual H-H formalism and the instantaneous current. Standard whole-cell
method. Rs, 5 M;
Cm, 6 pF; 80% compensated.
[View Larger Version of this Image (13K GIF file)]
Two types of BK currents are found in rat chromaffin cells (Solaro et
al., 1995). This is summarized in Figure
1A. Ca2+ (4 µM) was introduced directly into each of
two chromaffin cells using the standard whole-cell patch-clamp
technique. In one cell, a depolarizing step to +90 mV resulted in a
rapidly inactivating BK current (which we have termed
BKi current, resulting from
BKi channels). In the other cell, the step to +90
mV activated a sustained current (which we have termed
BKs current, resulting from
BKs channels). This sustained current is more
reminiscent of BK currents generally found in most other cell types.
BKi current is found in ~75% of rat chromaffin
cells; the remaining cells show either pure BKs
current or a mix of BKi/BKs
current (Solaro et al., 1995). In this paper, most results use cells
with BKi current. In part, this reflects the
greater likelihood of obtaining cells with BKi
current, but also, as will be shown below, certain features of
BKi current are particularly useful for answering
some of the questions considered here. To provide assurance that our
conclusions do not arise from some novel aspect of
BKi current, the behavior of
[Ca2+]i in cells with
BKs current also is presented in some cases.
Fig. 1.
Properties of BK currents found in rat chromaffin
cells. A, Examples of BKi and
BKs current traces recorded with pipettes
containing 4 µM are shown. Each cell was held
at a holding potential of 60 mV and stepped to +90 mV for 400 msec.
The voltage step evoked BKi current in the cell
shown on the left and BKs current in
the cell shown on the right. B,
BKi currents were recorded with pipettes
containing 1, 4, or 20 µM
[Ca2+]i, respectively, at
various voltages. Each cell was held at 60 mV and stepped from the
holding potential to potentials ranging from 30 to +90 mV in
increments of 10 mV. In all cases, the cell was bathed in an external
saline containing 0 [Ca2+]o. C,
The time constant of inactivation obtained by fitting current decay
with a single exponential is plotted as a function of
[Ca2+]i. Each point is
the mean (± SEM) of data collected from 4-6 cells. D, The
time constant of current inactivation is plotted against the membrane
potential at each
[Ca2+]i. Each point is
the mean (± SEM) of data collected from 4-6 cells. The dotted
lines in C and D indicate data from
excised patches reported earlier (Solaro and Lingle, 1992). Error bars
have not been indicated when smaller than symbol size.
[View Larger Version of this Image (26K GIF file)]
For any membrane current to be a useful assay of submembrane
[Ca2+]i, the dependence
on [Ca2+]i of some
parameter of channel function must be definable. Toward this goal, the
[Ca2+]i dependence of two
parameters of BK channel function, the conductance and the inactivation
rate, are defined first. Then this information is used to address four
questions: (1) What is the average
[Ca2+]i detected by BK
channels during depolarizing steps that produce
Ca2+ influx via Ca2+
channels? (2) Are there inhomogeneities in the
[Ca2+]i detected by BK
channels under such conditions? (3) What is the
[Ca2+]i detected by BK
channels during release of Ca2+ from internal
stores by muscarine? (4) What is the duration of submembrane
[Ca2+]i elevations with
short and long Ca2+ influx steps?
Ca2+ and voltage dependence of BK
current inactivation
The Ca2+ and voltage dependence of
BKi inactivation previously has been defined for
a limited set of [Ca2+]i
and voltages by using ensembles of channel openings from excised
patches (Solaro and Lingle, 1992). Because the present study relies on
the behavior of BK channels in whole-cell recordings, the
Ca2+ and voltage dependence of the inactivation
time constant was determined with the standard whole-cell technique,
using internal salines containing defined
[Ca2+]i (Fig.
1C,D). The previously published excised patch data are shown
for comparison also (Solaro and Lingle, 1992). The whole-cell
experiments extend the range of conditions
([Ca2+]i and voltage)
over which the inactivation time constant is defined and indicate that
the rate of inactivation becomes faster with increasing
[Ca2+]i and membrane
potential, as reported previously (Solaro and Lingle, 1992; Herrington
et al., 1995). Furthermore, the time constants from the whole-cell
experiments fall reasonably close to those determined from the patch
experiments, suggesting that any discrepancy between the expected and
the real [Ca2+]i at the
membrane during whole-cell dialysis is minimal and much smaller than
the changes that result from the various defined
[Ca2+]i solutions. The
usefulness of the inactivation time constant as an indicator of
submembrane [Ca2+]i is,
however, somewhat limited because it approaches its limiting value near
only 4 µM
[Ca2+]i at positive
voltages.
Ca2+ and voltage dependence of the fractional
activation of whole-cell BK current
Next, the Ca2+ and the voltage dependence of
BK conductance was characterized by introducing a saline with a defined
[Ca2+]i into the cell and
measuring the fractional activation of BK current at various potentials
in the absence of external Ca2+. Fractional
activation of BK current was determined by comparing the tail of BK
current elicited by a step to +90 mV after different activation
potentials to the total amount of BK current activated at +90 mV. The
+90 mV step was used because previous excised single-channel
experiments indicated that complete activation of BK current occurs at
this potential at [Ca2+]i
of 4 µM or higher (Solaro, 1995). Thus, current
at +90 mV provides a reasonable estimate of the maximum available
current at [Ca2+]i of 4 µM and above.
A sample current record obtained with 10 µM
[Ca2+]i and a
conditioning step to 5 mV is shown in Figure
2A. The trace shows that a large BK current
is turned on at +90 mV, with some current activation occurring during
the conditioning step. Careful examination of the trace also reveals
that there is an immediate, instantaneous increase in the current when
membrane potential is stepped from the conditioning potential to +90
mV. We will refer to this current as the ``instantaneous current''
(Fig. 2A). It arises because stepping the membrane potential
to +90 mV increases the driving force on the K+
ions moving through channels that are open at the conditioning
potential, thereby producing an ohmic and immediate increase in the
current. After the instantaneous increase in BK current, additional
activation of BK current occurs in accordance with the open probability
of the channels at +90 mV and the
[Ca2+]i present in the
cell. This produces a further increase in BK current, which
subsequently peaks and then falls as inactivation gains predominance.
We will refer to the maximum amplitude of the current at +90 mV as the
``peak current.''
Fig. 2.
Peak and instantaneous currents in
BKi cells. In A, 10 µM
[Ca2+]i was introduced
into the cell. First the cell was stepped from a holding potential of
60 to 5 mV for 20 msec and then stepped directly to +90 mV for 400 msec. After the step to 90 mV, there was a large, ohmic increase in
current (the instantaneous current), which was followed by some
additional transient activation of current. In B, currents
were recorded using pipettes containing 4, 10, 20, or 60 µM free
[Ca2+]i. The cells were
stepped from the holding potential (60 mV for cells with 4 and 10 µM
[Ca2+]i; 80 mV for
cells with 20 and 60 µM
[Ca2+]i) to either
65 (left traces) or to 5 mV (right
traces) for 20 msec and then stepped to +90 mV. The
instantaneous current at +90 mV became larger at the more positive
conditioning steps and increased further as the
[Ca2+]i was increased. In
all cases, cells were bathed in an external saline containing 0 [Ca2+]o.
Arrows on each trace denote the instantaneous (open
arrows) and peak (closed arrows) current.
Sampling period, 500 µsec.
[View Larger Version of this Image (13K GIF file)]
Because the open probability of BK channels at +80 mV is near maximal
at [Ca2+]i of 4 µM and above (Solaro, 1995), the peak current
at +90 mV is a reasonably good estimate of the maximum available BK
current at the time of the voltage step to +90 mV. Thus, the fractional
activation of BK current at the different conditioning potentials can
be determined simply by computing the ratio of the instantaneous to the
peak current (see Fig. 4). This ratio defines the fraction of channels
open at the conditioning potential compared with the total number of
channels that can be opened at +90 mV.
Fig. 4.
Fractional activation of BKi
current at various
[Ca2+]i and voltages. The
instantaneous current was normalized to the peak current activated at
+90 mV. This ratio represents the fractional activation of BK current
at various conditioning potentials. Each point represents the mean (± SEM) of 6-7 cells. Procedures for estimating the instantaneous current
and factors that may affect estimates of both peak and instantaneous
current are described in Results. Fractional activation values obtained
at each [Ca2+]i were fit
with a single Boltzmann function that included a voltage-independent
nonzero term. The voltage-independent term for each fit represents the
amount of non-BK current activated by the voltage step to +90 mV during
the first sampling interval. Fits were constrained to
Gmax of 1.0. The voltages of
half-activation (V50) and slope factors at
the various [Ca2+]i were
as follows: 4 µM
[Ca2+]i, 48.2 mV with a
slope factor of 13.1 mV; 10 µM
[Ca2+]i, 3.4 mV with a
slope factor of 15.3 mV; 20 µM
[Ca2+]i. 22.0 mV with a
slope factor of 14.1 mV; and 60 µM
[Ca2+]i, 41.6 mV with a
slope factor of 15.8 mV. The dotted lines indicate the
fractional activation data obtained by fitting each current trace with
a modified Hodgkin-Huxley function and estimating the peak current
from the fit, as described in Materials and Methods.
[View Larger Version of this Image (30K GIF file)]
What are the possible errors in this approach of computing the
fractional activation of BK current? One error may arise from imprecise
estimates of the instantaneous current. This could result from two
factors: the finite current sampling rate and the finite settling time
of the imposed voltage change. In the first case, the magnitude of
instantaneous current may be overestimated because of BK current
activation during the finite sampling period. In the second case, early
current points after a voltage step may reflect charging of cell
capacitance and slow changes in the command voltage resulting from
series resistance limitations. Because many of the experiments
described in this paper required voltage protocols with sampling rates
as slow as 500 µsec, the impact of these factors on estimates of
instantaneous current are summarized in Figure 3 for the
two types of whole-cell recording conditions. Currents were digitized
at either 50 or 500 µsec. At typical Rs
and Cm for cells in this study, voltage
settling times are ~250-300 µsec in cells clamped with the
perforated-patch method and ~75-100 µsec for standard whole-cell
recordings. Thus, in perforated patch-clamped cells, the new command
potential is not imposed fully for ~250-300 µsec, and, in general,
the first sampled point 500 µsec after the initiation of the voltage
step is essentially identical to the estimate of instantaneous current
obtained by sampling at 50 µsec intervals (Fig. 3Aa). In
contrast, in standard whole-cell recordings, current through open
channels seems to sense fully the newly imposed voltage within ~100
µsec (Fig. 3Ab). Once the new command voltage is imposed,
relatively smaller changes in BK current occur during the subsequent 50 µsec sampling periods, and the ohmic component through BK channels is
apparent as the first point after the termination of rapid change in
current. Thus, for standard whole-cell recordings, the current measured
at 500 µsec after the voltage step overestimates the true amplitude
of the instantaneous current.
To apply a correction for this sampling error, the following procedure
was used. The initial phase of BK current activation is described well
by a simple exponential function (Solaro et al., 1995). Therefore, BK
current was assumed to activate in a primarily linear manner during the
first few milliseconds after a voltage step. The measured current
values at 500 and 1 msec were used, therefore, to calculate the amount
of current that would have been observed at 250 µsec, assuming this
linear rate of current activation. The 250 µsec extrapolation point
was used because the amplitude of the instantaneous current defined at
this time point agreed well with that obtained by directly sampling at
50 µsec intervals. Thus, fractional activation in cells dialyzed with
defined [Ca2+]i salines
was obtained by taking the ratio of the corrected instantaneous current
and the measured peak current.
A second problem arose from the fact that the peak current would be
somewhat underestimated by inactivation that might occur between the
voltage step and the time of peak current. We attempted to estimate the
extent of this inactivation by fitting current traces with a modified
Hodgkin-Huxley model (1952) of current activation-inactivation, as
described in Materials and Methods (Fig. 3B). The peak
current given by this fit may somewhat overestimate the true peak
current, because this model assumes independent activation and
inactivation gating, whereas inactivation gating in
BKi channels is coupled partially to activation
gating (Solaro and Lingle, 1992). The fitting procedure, therefore,
places an upper limit on the estimate of the true peak current. The
voltages of half-activation (V50s) obtained
by using amplitude estimates derived from this fitting procedure were
shifted to the right by ~15 mV at 4 µM
[Ca2+]i, 5 mV at 10 µM
[Ca2+]i, and by smaller
values (1-2 mV) at 20 and 60 µM
[Ca2+]i (Fig.
4). These shifts in V50 at any individual
[Ca2+]i were small compared with the
differences in V50 among different
[Ca2+]i. Any errors
resulting from incorrect estimates of the true peak current are
therefore small. Thus, when comparing the calibration data to
fractional activation data produced by physiological
[Ca2+]i elevations, we
used only the directly measured peak current values.
A final potential source of error arises from contamination of
the measured currents with the voltage-dependent,
Ca2+-independent K+
current. The amplitude of this current in rat chromaffin cells is
typically ~1-1.5 nA at +90 mV so that contamination of the estimate
of both the instantaneous and peak currents is ~5-8%. An error of
this magnitude is substantially less than the differences that result
from the various defined
[Ca2+]i solutions.
The above considerations were applied in determining the fractional
activation of BK current at various
[Ca2+]i and voltages.
Resulting fractional activation curves are shown in Figure 4. The
procedure yielded roughly similar conductance-voltage curves for both
BKi and BKs currents
(Solaro et al., 1995). The values for the voltages of half-activation
(V50) are within the range of estimates of
V50 obtained from single BK channels in
excised chromaffin cells patches (Solaro, 1995). Qualitatively, the
results indicate that, at ~0 mV, submembrane
[Ca2+]i must exceed 60 µM to result in maximal BK current activation,
whereas at 4 µM
[Ca2+]i there is minimal
activation of BK current. At voltages exceeding +80 mV, BK current is
activated maximally at
[Ca2+]i of 4 µM or above. At 1 µM
[Ca2+]i, BK current is
activated detectably, although only at very positive potentials (Fig.
1B). Finally, at 500 nM
[Ca2+]i, virtually no BK
current activation can be seen even at +90 mV (data not shown).
Fractional activation of BK current during depolarization-elicited
Ca2+ influx
To use the fractional activation data of Figure 4 to assay the
[Ca2+]i detected by BK
channels during Ca2+ influx, the maximal
activatable BK current must be defined for each cell. Therefore, the
instantaneous and peak currents elicited by Ca2+
influx in cells clamped in the perforated patch-clamp configuration
were characterized. Figure 5A shows traces
from a cell in which currents activated during a voltage step to +81 mV
are compared with and without a 200 msec conditioning step to +1 mV.
Test pulses to +81 mV were used, because there is minimal
Ca2+ influx at this potential and BK currents can
be viewed with minimal contamination from other currents. Because of
the Ca2+ influx that occurs during the loading
step to +1 mV, BK current activated during the step to +81 mV greatly
exceeds the purely voltage-dependent K+ current
activated by stepping directly to +81 mV. The instantaneous current
after the Ca2+ loading step indicates that a
substantial number of BK channels are open at this time. Examination of
the fractional activation curves in Figure 4 indicates that, at +1 mV,
submembrane [Ca2+]i must
be in excess of 4 µM to produce an
instantaneous current of this magnitude. Now, if the maximum number of
BK channels that can be activated by the step to +81 mV can be defined,
it would provide a way of more precisely defining the average
[Ca2+]i detected by BK
channels during Ca2+ influx.
Fig. 5.
Effect of varying the duration of the
Ca2+ influx step on instantaneous and peak
currents. A, Outward current in a perforated patch-clamped
cell is shown with and without Ca2+ influx. The
cell was held at 69 mV and stepped to +81 mV with or without a
conditioning step to +1 mV. On the left, the direct step to + 81 mV activates only Ca2+-independent,
voltage-dependent K+ current. On the
right, the step to +1 mV produces Ca2+
influx and results in activation of BK current at +81 mV. Note the
prominent instantaneous current at +81 mV. Closed and
open arrows indicate peak and instantaneous currents,
respectively. B, The variation of peak and
instantaneous currents with increasing influx is demonstrated in a cell
with BKi current. The cell was held at 69 mV,
stepped to 9 mV to produce Ca2+ influx, and
then stepped to +81 mV. The duration of the influx step was varied from
0 to 140 msec in increments of 10 msec. The resulting current traces
are shown on the left, and the instantaneous
(squares) and peak (circles) current amplitudes
at +81 mV for each Ca2+ influx step are plotted
against the duration of the influx step on the right. Only
every other current trace is shown for clarity. Perforated-patch
method. Rs, 9.5 M;
Cm, 5.5 pF; 80% compensated. C,
Same as B, but in a cell expressing
BKs current. Perforated-patch method.
Rs, 14 M;
Cm, 5 pF; 80% compensated. Sampling
period, 500 µsec in all cases.
[View Larger Version of this Image (24K GIF file)]
To define the maximal BK current that can be activated at +81 mV,
Ca2+ influx was increased progressively by
increasing the duration of a 9 mV Ca2+ loading
step in an attempt to load the cell with Ca2+
sufficiently so as to activate all BK channels (Fig. 5B).
The resulting behavior of the instantaneous and peak currents shows
several features worth noting. (1) Even the short duration
Ca2+ influx steps produce a clear, instantaneous
current. The peak current at +81 mV with such small influx steps is
almost all instantaneous current. (2) Small increments in the duration
of Ca2+ influx increase both the peak and the
instantaneous currents. However, as the duration of the influx step is
increased further (influx steps >30-40 msec), the instantaneous
current plateaus, whereas subsequent increases in
Ca2+ influx increase only the peak current.
Eventually (typically after 100-300 msec of Ca2+
influx), the peak current also reaches a maximal level (Fig.
5C). (3) After long (typically 100 msec)
Ca2+ loading steps, rates of inactivation of
BKi current at +81 mV increase to values that
suggest the residual submembrane
[Ca2+]i is equal to or
above 3-5 µM (Solaro et al., 1995). The
fractional activation data of Figure 4 indicate that this
[Ca2+]i should be
sufficient to produce maximal BK conductance
(gmax) at +81 mV. (4) With even longer
Ca2+ loading steps, both the instantaneous and
the peak current amplitude of BKi current begin
to decrease. This decrease reflects inactivation of
BKi channels at the potential of the
Ca2+ loading step, as reported previously (Solaro
et al., 1995).
Figure 5C shows the effect of varying the
Ca2+-influx duration on the instantaneous and the
peak current in a cell with BKs current. The
basic behavior of the BKs current is similar to
BKi current. However, in contrast to
BKi current, the instantaneous current in
BKs cells consistently shows a tendency to
increase slowly with the loading-step duration. This point is discussed
later.
Does the maximal peak current provide an estimate of the maximal
available current at the time of the +81 mV voltage step? This was
tested in two ways. First, Ca2+ influx through
Ca2+ channels was increased by changing the
extracellular [Ca2+] from 1.8 to 9 mM to test whether increases in
Ca2+ influx result in the additional recruitment
of any ``silent'' BK channels that are not active in the 1.8 mM
[Ca2+]o saline (Fig.
6A). In 9 mM
[Ca2+]o, the
instantaneous current amplitude elicited by short influx steps is
increased relative to the current elicited in 1.8 mM
[Ca2+]o. However, the
maximal peak current amplitudes are similar in both cases, suggesting
that all of the BK channels that are activated at +81 mV in the 9 mM
[Ca2+]o saline also can
be activated in the 1.8 mM
[Ca2+]o saline, albeit at
longer Ca2+ influx steps. Second, in three cells,
the effect of an additional depolarization to +111 mV after the +81 mV
step was tested. After Ca2+ influx steps of 200 msec or longer, this depolarization resulted in no additional increase
in conductance over that observed at +81 mV (Fig. 6B),
indicating that maximal conductance had been achieved. It is worth
noting that because substantial inactivation of BK current occurs in
many cells during the Ca2+ loading step, the
maximal peak current at +81 mV will not represent all the BK
current in a given cell but, rather, the activation of all
available BK current.
Fig. 6.
The maximal peak current elicited at +81 mV with
long Ca2+ influx steps defines the maximal
available BK conductance. A, The cell was stepped from the
holding potential (69 mV) to 9 mV to produce
Ca2+ influx and then stepped to +81 mV. The
top traces show currents elicited in external saline
containing 1.8 mM
[Ca2+]o, whereas the
bottom traces show currents in the same cell in external
saline containing 9 mM
[Ca2+]o. On the
right, the instantaneous and peak currents are plotted
against the duration of the influx step. In both salines, the maximal
peak currents at +81 mV reach the same amplitude. Perforated-patch
method. Rs, 15.5 M;
Cm, 7.8 pF; 80% compensated. B,
The cell was stepped to 9 mV for varying durations to produce
Ca2+ influx and then stepped to +81 mV for 50 msec to activate the BK current robustly. After 50 msec at +81 mV, the
cell was stepped to +111 mV to detect the extent of BK current
activation at +81 mV. External Na+ was replaced
with N-methyl-D-glucamine (NMG) to
circumvent the effects of intracellular Na+ block
of BK channels at positive voltages (Yellen, 1984; see Materials and
Methods). After short Ca2+ loading steps, there
is some additional activation of BK current at +111 mV beyond what is
seen at +81 mV. However, after Ca2+ influx steps
of 150 msec and longer, additional activation at +111 mV does not
occur, indicating that maximal activation of BK current has been
achieved at +81 mV. Perforated-patch method.
Rs, 11 M;
Cm, 4.5 pF; 80% compensated. Sampling
period, 500 µsec.
[View Larger Version of this Image (20K GIF file)]
The fractional activation of BK channels activated by
Ca2+ influx steps long enough to produce maximal
activation of BK current was determined by using the above approach in
perforated patch-clamped cells at influx potentials in the 20 to +15
mV range (Fig. 7). Then these data were compared with
the calibration data in Figure 4. The comparison showed that, after
Ca2+ influx steps of long duration (100-400
msec), the average submembrane
[Ca2+]i in different
cells varies over a wide range, from somewhat under 10 µM in some cells to over 20 µM in others. However, in the majority of the
cells, the average submembrane
[Ca2+]i is ~10
µM.
Fig. 7.
The fractional activation of
BKi current elicited by large
Ca2+ influx steps corresponds to an average
submembrane [Ca2+]i of
10-20 µM. Ca2+ influx in
perforated patch-clamped cells was elicited by loading steps between
20 and +11 mV, which were followed by steps to +81 mV. In each cell,
the duration of the Ca2+ loading step was
increased progressively by using the protocol shown in Figure 5. The
fractional activation of BK current for a given
Ca2+ influx potential was measured by calculating
the ratio of the instantaneous to the peak current for the trace
showing the maximal peak BK current. In some cases, traces for which
the Ca2+ loading-step durations were longer than
the duration that elicited the maximal peak BK current were used. The
fractional activation data obtained at various
Ca2+ influx potentials from 34 cells are shown
here. Solid lines are the fractional activation
calibration curves from Figure 4.
[View Larger Version of this Image (22K GIF file)]
BK channels close to Ca2+ channels can be unmasked
by EGTA
Although the above result indicates that the average
[Ca2+]i detected by
individual BK channels at the end of long Ca2+
influx steps is ~10 µM, it does not rule out
large variations in the
[Ca2+]i detected by
individual BK channels arising from differences in the distances
between BK and Ca2+ channels. Thus, BK channels
located close to Ca2+ channels would experience
high [Ca2+]i, and those
located at a distance, low
[Ca2+]i. If this is true,
the exogenous Ca2+ buffers, EGTA and BAPTA, might
differentiate between those BK channels close to
Ca2+ channels and those more distant. The
equilibrium Ca2+ affinities of these two buffers
are comparable, but because the Ca2+ binding rate
of EGTA is ~100 times slower than BAPTA, EGTA should be unable to
buffer [Ca2+]i as
effectively as BAPTA in the immediate vicinity of
Ca2+ channels (Neher, 1986; Stern, 1992). Thus,
if BK channels are coupled closely to Ca2+
channels, EGTA should be less effective than BAPTA in preventing their
activation during influx. This approach has been used in past studies
to demonstrate coupling between Ca2+ and BK
channels in the frog hair cell (Roberts, 1993) and between
Ca2+ channels and secretory vesicles in the
terminals of the squid giant axon (Adler et al., 1991).
BK currents were activated with intracellular EGTA in the range of 80 µM to 5 mM, using the
standard whole-cell technique (Fig.
8A). In 80 µM
EGTA, the behavior of the instantaneous and peak current elicited by
Ca2+ loading steps of different durations
primarily approximates what is observed in cells studied with the
perforated patch-clamped method. Instantaneous current reaches a
plateau, whereas the peak current slowly increases with loading-step
duration and then decreases as inactivation accumulates at 9 mV.
However, at higher concentrations of EGTA, the results differ. At 400 µM EGTA, peak current exhibits an interesting
biphasic behavior, decreasing at first but then rebounding as the
influx step duration is increased further. The instantaneous current,
however, shows only a steady decrement under these conditions. At 1 mM EGTA, almost all of the current activated at
+81 mV at all Ca2+ loading steps is instantaneous
current. This current becomes smaller as the influx duration is
increased. At 5 mM EGTA, also, almost all of the
peak current elicited with both the short and long influx steps is
instantaneous current, which rapidly becomes attenuated as the influx
step duration is increased. The average instantaneous current recorded
with 5-20 msec voltage steps to 9 mV was 5253 ± 548 pA in
perforated patch-clamped cells (n = 17), 5147 ± 642 pA
with 80 µM EGTA (n = 10), 5041 ± 908 pA with 1 mM EGTA (n = 7),
and 5833 ± 1293 pA with 5 mM EGTA
(n = 5). Thus, about the same amount of BK current is
activated by brief Ca2+ influx steps irrespective
of the presence or absence of EGTA. In contrast, BAPTA at
concentrations of 1 and 5 mM completely blocked
activation of BK current, whereas 400 µM BAPTA
eliminated most BK current (Fig. 8B).
Fig. 8.
EGTA eliminates the slowly developing increase in
peak current but not the instantaneous current, whereas BAPTA
eliminates all BK current. A, EGTA at concentrations ranging
from 80 µM to 5 mM was
introduced into cells, and BK current was elicited with
Ca2+ influx. Cells were held at 69 mV, stepped to 9 mV
to produce Ca2+ influx, and then stepped to +81
mV. The duration of the step to 9 mV was increased from 0 to 220 msec
in increments of 20 msec. Shown below are the peak (circles)
and the instantaneous (squares) currents plotted against the
duration of the Ca2+ influx step. The first
voltage step was ignored in this plot, because it does not result in
any Ca2+ influx. The dotted line
in each case indicates the amplitude of the steady-state
Ca2+-independent, voltage-dependent current at
+81 mV. As the EGTA concentration is increased, the slowly developing
rise in peak current is eliminated. B, BAPTA at
concentrations of 400 µM and 1 and 5 mM was introduced into each of three chromaffin
cells. BAPTA even at 400 µM is quite effective
in eliminating BK current. Note the different y-axis scales
in A.
[View Larger Version of this Image (29K GIF file)]
What do these results indicate? EGTA at concentrations of 1 mM or above abolishes the slowly developing
increase in peak current seen in perforated patch-clamped cells (Fig.
5) and in cells dialyzed with 80 µM EGTA (Fig.
8A). However, it does not abolish the instantaneous
current elicited with short influx steps, suggesting that the BK
channels producing this current are located sufficiently close to
Ca2+ channels so as to be unaffected by the
buffering action of EGTA. The close proximity to
Ca2+ channels probably leads to high
[Ca2+]i around these BK
channels, producing their rapid inactivation and leading to attenuation
of the instantaneous current as the influx step duration is increased.
In contrast, the gradual increase in the peak current seen in
perforated patch-clamped cells (Fig. 5B) and with 80 µM EGTA (Fig. 8A) probably
reflects the activation of BK channels at increasing distances from
Ca2+ channels. The substantial time-dependent
activation of this current at the long influx durations at +81 mV
implies that the average open probability of these BK channels is low
and suggests that they experience relatively low
[Ca2+]i.
The current traces at 400 µM EGTA (Fig.
9A) lend particular support to the idea of
nonuniformity in the
[Ca2+]i detected by
different BK channels. This EGTA concentration seems optimal to produce
a clear separation between the fall of the early instantaneous BK
current and the slowly developing peak current. The rapid and severe
inactivation of the early instantaneous current evident at the longer
influx durations suggests that the BK channels producing this current
are exposed to high
[Ca2+]i. In contrast, the
substantial time- and voltage-dependent activation of BK current at +81
mV over a period when the instantaneous current is abolished almost
completely suggests that the BK channels contributing to the slowly
developing current must be exposed to lower
[Ca2+]i. The biphasic
behavior of the peak current observed in cells with
BKi current is not seen in cells with
BKs current (Fig. 9B), emphasizing
that it is a consequence of the inactivation behavior of
BKi channels.
Fig. 9.
400 µM EGTA uncovers BK
channels closely associated with Ca2+ channels
from those at a distance. A, Two examples of
BKi currents recorded in the presence of 400 µM EGTA in the cell are shown. The voltage
protocol is the same as that in Figure 8. Amplitudes of the peak
(circles) and instantaneous (squares) currents
plotted against the duration of the influx step are shown on the
right. The dotted line in each case
indicates the amplitude of the steady-state
Ca2+-independent, voltage-dependent current in
each cell. In each case, the instantaneous current becomes
progressively smaller as the duration of the influx step is increased.
The peak current becomes smaller in the first few influx steps but then
recovers as the influx step duration is still increased. B,
The behavior of BKs current with 400 µM EGTA in the cell is shown.
Instantaneous current is more or less steady, and peak current
increases with increasing influx duration.
[View Larger Version of this Image (38K GIF file)]
In sum, the above results indicate that some fraction of BK channels in
chromaffin cells are sufficiently close to Ca2+
channels to be unaffected by EGTA. Furthermore, when EGTA is used to
unmask these BK channels, Ca2+ influx steps with
durations >50 msec progressively and fully inactivate them. This rapid
inactivation is consistent with the hypothesis that these BK channels
are activated by relatively high
[Ca2+]i. In contrast, in
perforated patch-clamped cells, the instantaneous current always
maintains a plateau, with only a small decrement after longer
Ca2+ influx steps. The unmasking of the
inactivation of some BK channels in the presence of high EGTA suggests
that, in normal cells, the apparent plateau really reflects a balance
of inactivation of BK channels closest to sites of
Ca2+ influx and additional activation of channels
just outside of these regions. Thus, the plateau may reflect a changing
population of active BK channels. We envision the population of BK
channels contributing to the instantaneous current in
BKi cells as an annulus around points of
Ca2+ influx that moves outward as the proximal
channels become inactivated during the long influx steps. Furthermore,
as the Ca2+ loading-step duration is increased,
the instantaneous component would arise from a progressively larger
population of BK channels sensing, on average, a progressively lower
submembrane [Ca2+]i. In
BKs cells, on the other hand, with longer
Ca2+ influx steps the activation of more distant
BK channels simply results in a gradual increase in the size of the
instantaneous current, as noted earlier (Fig. 5C).
Submembrane [Ca2+]i elevations caused
by intracellular Ca2+ release
Rat chromaffin cells display robust muscarinic acetylcholine
receptor (mAChR)-mediated release of Ca2+ from
intracellular stores, presumably via an
IP3-mediated process (Neely and Lingle, 1992b;
Herrington et al., 1995). The time course of the
Ca2+ elevation can be monitored by repeated steps
to +81 mV to activate BK current during the application of muscarine
(Herrington et al., 1995). Using this protocol in cells with
BKi current, large inactivating BK currents are
seen during the muscarinic response, which typically lasts ~15-20
sec (Fig. 10). At the peak of the muscarinic response,
the inactivation time constants of BKi currents
are typically 40-60 msec (Herrington et al., 1995; Solaro et al.,
1995). Based on inactivation time constants from Figure 1, the
implication is that the submembrane
[Ca2+]i reaches 3-5
µM during the peak of the response. Analysis of
the rates of inactivation of successive traces during the peak of the
[Ca2+]i elevation also
shows that [Ca2+]i
remains elevated above 1 µM for several seconds
(typically 4-6 sec with 15 sec 50 µM muscarine
applications). Thus, during each of the 400 msec +81 mV voltage steps,
it is unlikely that there are dramatic temporal variations in the
[Ca2+]i. Instead,
submembrane [Ca2+]i seems
to remain fairly constant during these steps.
Fig. 10.
Ca2+ influx elevates the
average submembrane
[Ca2+]i to higher levels
than does mAChR activation. A, A perforated patch-clamped
cell was held at 69 mV and stepped repeatedly to +81 mV for 400 msec
every 1.25 sec. After 5 sec, muscarine (50 µM)
was applied for a period of 20 sec. Outward currents elicited before,
during, and after the muscarine application are shown. For clarity, not
all traces have been plotted. The peak current in each trace is plotted
against time in the bottom plot. mAChR stimulation results
in the release of Ca2+ from intracellular stores,
which elicit large, inactivating BK currents here. Note that after the
first surge of [Ca2+]i,
BK currents are suppressed briefly before rebounding. This is
attributable to Ca2+-induced inactivation of BK
current and its subsequent recovery as
[Ca2+]i levels fall
(Herrington et al., 1995). Perforated-patch method.
Rs, 13 M;
Cm, 6 pF; 80% compensated. B,
The [Ca2+]i elevation
resulting from mAChR activation is compared directly with that
resulting from influx in the same cell. Top trace
shows BK current elicited in normal external saline with a 25 msec
influx step to 9 mV, followed by a 400 msec step to +81 mV.
Bottom trace shows current elicited during a muscarine
response from the same cell in a zero external
[Ca2+] saline with the same voltage protocol
applied every 1.25 sec. Note that the instantaneous current elicited
with Ca2+ influx is significantly larger than
that produced by mAChR stimulation, but the peak current is smaller and
the rate of inactivation is slower. Closed and open
arrows indicate the peak and instantaneous current,
respectively. Perforated-patch method. Rs,
14.4 M; Cm, 7 pF; 80% compensated.
Sampling period, 500 µsec.
[View Larger Version of this Image (22K GIF file)]
We compared the submembrane
[Ca2+]i elevation
resulting from mAChR activation with the elevation caused by
Ca2+ influx. The consequences of influx are
demonstrated by the top trace in Figure 10B, in which the
cell was stepped to 9 mV for 25 msec to elicit influx and then
stepped to +81 mV. Ca2+ influx resulted in the
typical activation of BK current, and, after a step to +81 mV, there
was the usual instantaneous current with some additional slow current
activation. Then external Ca2+ was removed, and
50 µM muscarine was applied to the cell, during
which the previous voltage protocol was applied every 1.25 sec. At the
peak of the muscarine-induced
[Ca2+]i elevation, the
step to 9 mV resulted in little detectable activation of BK current
and little instantaneous current at +81 mV. However, a large, slowly
activating current was observed that inactivates more rapidly (time
constant = 48 msec) than the current resulting from
Ca2+ influx (time constant = 69 msec). These
inactivation rates indicate that, after termination of the 25 msec
influx step, the average submembrane
[Ca2+]i quickly falls
below the [Ca2+]i
persisting during mAChR-induced submembrane
[Ca2+]i elevation.
However, the instantaneous current of the muscarine-induced response is
significantly smaller than the instantaneous current resulting from
influx. This indicates that, during the step to 9 mV, BK
channels sense a higher average submembrane
[Ca2+]i when the source
of Ca2+ is influx through
Ca2+ channels rather than release from
intracellular stores.
These results argue that the mAChR-induced
[Ca2+]i elevation is
quite uniform over most of the response. Based on inactivation rates,
release of intracellular Ca2+ by mAChR activation
elevates the average submembrane [Ca2+] to ~4
µM, yet little or no BK current activation
occurs at 9 mV during the muscarine response. Because detectable
instantaneous current after steps to +81 mV would be expected if even
10% of BK channels were exposed to 10 µM
[Ca2+], the previous result suggests that few,
if any, BK channels are exposed to [Ca2+] as
high as 10 µM. Furthermore, at the peak of the
response to muscarine, steps from +81 to +111 mV produce no additional
time-dependent activation of BK current (data not shown), suggesting
that there are very few BK channels exposed to
[Ca2+] <4 µM. This
implies that the mAChR-induced submembrane
[Ca2+]i elevation may
affect a significant portion of the membrane, with the minimal and
maximal submembrane
[Ca2+]i probably varying
less than two- to threefold.
Short Ca2+ influx steps activate some BK channels with
near-maximal open probabilities
Results obtained with high concentrations of exogenous buffers
(Figs. 8, 9) suggest that the instantaneous current at +81 mV after
short depolarizing steps to 9 mV arises from a fraction of
BKi channels seeing a high
[Ca2+]i, whereas the
instantaneous current resulting from long influx steps arises from a
population of channels seeing, on average, a lower
[Ca2+]i. In the former
case, if the BK channels underlying the instantaneous current see
sufficient [Ca2+]i so as
to be maximally or near-maximally activated at 9 mV, an
additional increment in cytosolic
[Ca2+]i should have
little or no effect on the amplitude of the instantaneous current. In
contrast, the same additional increment in cytosolic
[Ca2+]i should increase
the instantaneous current resulting from long influx steps, in which
the average [Ca2+]i is on
the order of 10 µM (Fig. 7). This hypothesis
was tested by using the elevation of
[Ca2+]i by mAChR
activation as a means to produce the additional
[Ca2+]i increment. As
argued earlier, mAChR activation produces a relatively uniform
submembrane [Ca2+] of ~3-5
µM. Furthermore, no BK channels detect
[Ca2+]i sufficient to
produce substantial activation at 9 mV.
BK current evoked by muscarine and a 5 msec Ca2+
influx step were compared to the current evoked by the
Ca2+ influx step alone. The 5 msec depolarization
to 9 mV resulted in an instantaneous current of 4355 pA (Fig.
11A). Subsequent mAChR activation
immediately before the next depolarizing step elevated submembrane
[Ca2+]i further but
produced little change in the instantaneous current, yet the rate of BK
current inactivation at +81 mV placed submembrane
[Ca2+]i near 4 µM. This suggests that the BK channels
activated during the short influx step already are sensing a
[Ca2+]i of at least 60 µM. In contrast, after long influx steps, mAChR
activation immediately before the influx step consistently enhances the
instantaneous current. For instance, as shown in Figure 11B,
after a 50 msec influx step, the instantaneous current increased from
4121 to 7119 pA. Assuming that the current elicited at +81 mV with
mAChR activation is the maximal BK current available for activation,
the fractional activation of BK channels at 9 mV increased from 0.31 to 0.54, indicating an increase in submembrane
[Ca2+]i from 10 µM to ~15 µM (from
Fig. 4). This change in
[Ca2+]i is consistent
with the addition of the ~4 µM
[Ca2+]i that normally
results from mAChR activation (Fig. 10). Essentially identical results
were obtained in four cells with a 0.25% average increase in
fractional activation after a short (5-10 msec) influx step and a 57%
average increase after a long (50-100 msec) influx step. Together with
the EGTA data (Fig. 8), these results suggest that the BK channels that
contribute to the instantaneous current during short
Ca2+ influx steps sense average submembrane
Ca2+ concentrations of at least ~60
µM, whereas BK channels that contribute to the
instantaneous current with long influx steps sense average submembrane
Ca2+ concentrations closer to 10 µM.
Fig. 11.
The average fractional activation of open BK
channels after short depolarizations is higher than the average
fractional activation of open BK channels after long depolarizations.
A, BK current was elicited in a perforated patch-clamped
cell with a step to 9 mV for 5 msec to produce
Ca2+ influx and immediately followed by a step to
+81 mV. The voltage protocol was repeated every 1.5 sec, and,
immediately after the second episode, 50 µM
muscarine was applied. Shown here are the current traces immediately
before and after the muscarine application. Instantaneous and peak
current before muscarine application were 4355 and 5097 pA.
Instantaneous and peak current after muscarine application were 4121 and 13232 pA. B, The Ca2+ influx step
was increased to 50 msec in the same cell. As before, 50 µM muscarine was applied after the second
episode. Shown here are the current traces immediately before and after
muscarine application. Instantaneous and peak currents before muscarine
application were 4414 and 7900 pA. Instantaneous and peak currents
after muscarine application were 7119 and 12138 pA. Closed
and open arrows indicate peak and instantaneous
currents, respectively. Perforated-patch method.
Rs, 14 M;
Cm, 6 pF; 80% compensated. Sampling
period, 500 µsec.
[View Larger Version of this Image (10K GIF file)]
What fraction of BK channels is sufficiently close to
Ca2+ channels to be maximally activated by the
short influx steps? To determine this, the instantaneous current at +81
mV activated by short Ca2+ influx steps was
compared with the total BK current in the cell. Total BK current was
determined roughly by steps to +81 mV during mAChR-induced
[Ca2+]i elevation.
Inactivation before the +81 mV step was minimized by holding at
negative potentials before stepping to +81 mV, and the amount of
inactivation during the rising phase of the current at +81 mV was
accounted for by fitting the current with the modified Hodgkin-Huxley
function described in Materials and Methods. Using this procedure, in
15 cells the instantaneous current activated at +81 mV after 5-10 msec
depolarizations to 9 mV involved 17.7 ± 6.7% of the total
population of BK channels. If the maximal BK current were really 50%
higher, the fraction of BK channels exposed to
[Ca2+] of 60 µM or
higher would be 11.9 ± 4.5%.
Clearance of Ca2+ after influx
The persistence of submembrane
[Ca2+]i after the
termination of Ca2+ influx was studied by evoking
Ca2+ influx with depolarizations of different
durations. Then the amount of BK current activation that can occur
after a variable recovery period after the termination of the influx
step was determined (Fig. 12). This protocol showed
that, with very small Ca2+ influx steps,
submembrane [Ca2+] fell very rapidly; a step to
+81 mV 5 msec after the termination of Ca2+
influx resulted in no detectable BK current activation (Fig.
12A). However, as the influx step was made longer,
residual BK current activation was seen at progressively later time
points after the termination of Ca2+ influx. With
Ca2+ influx steps of 50 msec or longer, BK
current activation often was seen hundreds of milliseconds after
the termination of the influx step.
Fig. 12.
Submembrane
[Ca2+]i drops rapidly
after short influx steps but persists at significant levels for long
durations after long influx steps. The cell was held at 69 mV,
stepped to 9 mV to produce Ca2+ influx, and
then stepped back to 80 mV for variable recovery periods before
applying a step to +81 mV to activate BK current again. The recovery
period duration at 80 mV ranged from no recovery (0 msec) to several
seconds. In A, the influx step was 5 msec long; the recovery
steps at 80 mV were incremented by 5 msec. In B, the
influx step was 10 msec in duration; the recovery steps were
incremented by 10 msec. In C, the influx step was 100 msec
in duration; the recovery steps were incremented by 100 msec. In
D, the influx step was 400 msec; the recovery steps were
incremented by 200 msec. The inactivation time constants of BK currents
elicited at +81 mV in D are shown in the inset on the
right. Because channels recover from inactivation during the
initial recovery steps, large increases in BK current elicited at +81
mV are observed as the recovery duration is increased. Perforated-patch
method. Rs, 9 M;
Cm, 6 pF; 80% compensated.
[View Larger Version of this Image (25K GIF file)]
In fact, when the Ca2+ loading steps are
200 msec or longer, rates of BK current inactivation at +81 mV often
remain very fast even hundreds of milliseconds (200-400 msec) after
the termination of the influx step. For instance, in the cell shown in
Figure 12, the time constant of inactivation stays near its limiting
value, ~50 msec, for 200-300 msec after the termination of a 400 msec influx step. This indicates that the submembrane
[Ca2+] remained ~3-5
µM during this period. Longer periods of
recovery after termination of influx result in the slowing of the
inactivation rate, indicating a gradual fall in the submembrane
[Ca2+]i. What is
noteworthy is that even after long recovery times, e.g., 800 msec, the
rates of inactivation still correspond to a submembrane
[Ca2+]i above 1 µM. Note that the current elicited at +81 mV
increases initially as the recovery step at 80 mV is increased (Fig.
12D). This results from the recovery from inactivation of BK
channels that have inactivated during the Ca2+
loading step (Solaro et al., 1995).
Because detectable activation of BK current can occur at +81 mV with
[Ca2+]i as little as 1 µM (Fig. 1), the complete absence of BK current
after the shortest loading steps suggests that
[Ca2+]i falls from at
least 60 to <1 µM within 5 msec after the
termination of Ca2+ influx. In contrast, after
longer Ca2+ loading steps (>20 msec),
[Ca2+]i falls at rates that depend
on the magnitude of the Ca2+ load, although the
high [Ca2+]i in the
immediate vicinity of Ca2+ channels must still
drop rapidly to the average cytosolic
[Ca2+]i levels.
DISCUSSION
In this study, we determined the Ca2+
sensitivity of BK channels in chromaffin cells and then examined the
range of [Ca2+]i detected
by BK channels after Ca2+ influx and the release
of Ca2+ from internal stores. The key findings
follow. (1) During Ca2+ influx, ~10-20% of
the BK channels in the cell detect submembrane
[Ca2+]i probably as high
as 60 µM. (2) Although these BK channels are
associated closely with Ca2+ channels, most BK
channels in chromaffin cells must be at some appreciable distance from
Ca2+ channels. (3) The high concentrations of
Ca2+ in the vicinity of
Ca2+ channels fall rapidly after termination of
Ca2+ influx. (4) Release of intracellular
Ca2+ by mAChR stimulation produces a relatively
uniform submembrane
[Ca2+]i elevation of
~3-5 µM.
We have assumed that the Ca2+ and voltage
dependence of BK channel activation and inactivation are comparable in
perforated-patch recordings and under the conditions of our
calibration measurements involving standard whole-cell recordings. In
view of reports of BK channel modulation (Reinhart et al., 1991;
Twitchell and Rane, 1993; Bielefeldt and Jackson, 1994), this is an
assumption that will have to be addressed further. However, at present,
it is supported by the following observations. (1) After excision of
BKi channels in hundreds of inside-out patches,
we have never observed changes in inactivation properties or
sensitivity to [Ca2+]. (2) Properties of
BKi current in cells recorded with the
perforated-patch method are identical immediately before and after
activation of phospholipase C by mAChR activation (data not shown),
implying that there are no persistent effects on BK channels of any
mAChR-activated kinases. (3) The limiting BKi
inactivation rates are similar both in perforated-patch and whole-cell
recording, irrespective of the methods of
[Ca2+] elevation (Neely and Lingle, 1992b;
Herrington et al., 1995; Solaro et al., 1995). Although these are
indirect arguments, there is no observation that requires us to reject
this assumption.
Amplitudes of submembrane [Ca2+]i
elevations detected by BK channels
The results argue that the submembrane
[Ca2+]i elevations
resulting from Ca2+ influx are highly nonuniform.
Specifically, 5 msec after initiation of Ca2+
influx, ~10-20% of BK channels are exposed to
[Ca2+]i high enough to
produce maximal activation at 9 mV. This places the submembrane
[Ca2+]i detected by these
BK channels near 60 µM and possibly higher.
Because these channels are activated by short
Ca2+ influx steps rapidly and maximally, they are
positioned ideally to play a role in rapid repolarization of the
membrane potential during action-potential activity, a role previously
demonstrated for BKi current in rat chromaffin
cells (Solaro et al., 1995). The other 80% of BK channels are
activated only by prolonged Ca2+ influx steps and
detect lower [Ca2+]i.
With long influx steps that activate all available channels, the
average submembrane
[Ca2+]i approaches 10 µM. Because the channels in this latter
category are not exposed to
[Ca2+]i sufficient to
produce their rapid recruitment during single action potentials, it is
unlikely that they participate in the process of action-potential
repolarization. Instead, they might function under conditions that
involve Ca2+ release from intracellular
stores.
Spatial arrangement of BK and Ca2+ channels
BK channels in many cell types, including rat chromaffin cells,
require [Ca2+]i in excess
of 5 µM for activation at physiological
potentials (McManus, 1991), indicating that they have to be positioned
reasonably close to Ca2+ channels to see the
required [Ca2+]i.
However, this has been verified in only a few cell types. Among these,
the frog saccule hair cells (Roberts et al., 1990) and the presynaptic
terminals of the frog neuromuscular junction (Robitaille et al., 1993)
are reported to possess BK channels tightly colocalized with
Ca2+ channels. In the frog hair cell, this
colocalization is reported to lead to
[Ca2+]i in excess of 1 mM around the BK channels. Colocalization remains
less certain in hair cells from other species (Art et al., 1995). In
the neurons of Helix, only a subset of
Ca2+-dependent K+ channels
are coupled to Ca2+ channels (Gola and Crest,
1993), and, in bovine chromaffin cells, BAPTA is more effective than
EGTA in suppressing Ca2+-activated
K+ current (Marty and Neher, 1985), suggesting
that at least some of the Ca2+-activated
K+ channels are colocalized with
Ca2+ channels.
Possible distances between BK channels and Ca2+
channels can be calculated based on the published formalism for the
diffusion profile of Ca2+ away from single
Ca2+ channels (Neher, 1986; Stern, 1992).
Assuming single channel Ca2+ currents in the
range of 0.2-0.5 pA, these equations indicate that a BK channel
sensing [Ca2+]i of 60 µM must be 13-30 nm from the nearest
Ca2+ channel. Thus, the 10-20% of BK channels
in rat chromaffin cells that see
[Ca2+]i of 60 µM or higher are probably within this distance
from Ca2+ channels. However, it is important to
note that this estimate is critically dependent on the single channel
Ca2+ current value, a parameter subject to errors
in its estimation (Silberberg and Magleby, 1993). Despite this
uncertainty, a key question that arises is whether the BK channels
``close'' to Ca2+ channels are coupled to
Ca2+ channels by some specific mechanism or
whether an apparent ``coupling'' can arise simply from random
positioning of a large number of BK and Ca2+
channels in the membrane.
Two facets of the results are consistent with the view that the BK
channels exposed to the highest
[Ca2+]i may constitute a
distinct population coupled to Ca2+ channels by a
specific mechanism. First, the relative insensitivity of the
instantaneous current produced by the short Ca2+
influx steps over a wide range of EGTA concentrations suggests that the
BK channels underlying this current are not located at a
continuum of distances from Ca2+ channels. This
result is inconsistent with a scenario in which channels are
distributed randomly. Second, in the presence of 400 µM EGTA, BK currents exhibit an interesting
biphasic behavior (Figs. 8, 9), which may be explained most simply by
the presence of two populations of BK channels: one population
colocalized with Ca2+ channels that is activated
rapidly during Ca2+ influx and a second
population that is activated only with prolonged
Ca2+ influx and, presumably, located further away
from Ca2+ channels. In addition, simulations show
that a random distribution of BK and Ca2+
channels in the cell membrane is insufficient to account for 10-20%
BK channels detecting
[Ca2+]i of 60 µM or higher (Prakriya et al., 1996).
Clearance of Ca2+ after influx
The results show that the high
[Ca2+]i in the vicinity
of open Ca2+ channels must fall quite rapidly on
termination of Ca2+ influx. After very short
influx steps (5 msec) comparable to the widths of single-action
potentials (Solaro et al., 1995), BK channels that are activated almost
maximally during Ca2+ influx fail to be activated
by steps to +81 mV just 5 msec after the termination of
Ca2+ influx. This represents a
[Ca2+]i drop from at
least 60 to under 1 µM
almost two orders of
magnitude. Diffusion of Ca2+ from points of
Ca2+ entry can, in principle, account adequately
for this rapid fall in submembrane
[Ca2+]i. In contrast,
after Ca2+ influx durations of 50 msec or longer,
average submembrane
[Ca2+]i relax to under 1 µM over hundreds of milliseconds and even
seconds. The time course of this relaxation presumably reflects the
nature of Ca2+ sequestration and removal agents
such as mitochondria,
Na+/Ca2+ exchangers, and
Ca2+/ATPases. Mitochondria have been shown to
sequester Ca2+ during Ca2+
influx and gradually release it into the cytosol on termination of
influx (Werth and Thayer, 1994). Furthermore, there is recent evidence
that mitochondria are involved in shaping part of the
[Ca2+]i fall after very
long influx steps in rat chromaffin cells (Herrington et al., 1996;
Park et al., 1996).
Implications for secretion
Our results indicate that influx of Ca2+
raises the submembrane
[Ca2+]i to at least 60 µM in some locations, whereas release of
intracellular Ca2+ by mAChR activation raises
submembrane [Ca2+]i to
peak levels of only ~4 µM. Although the
absolute magnitude of the
[Ca2+]i elevations
produced by mAChR activation is less than that produced by influx, the
elevation produced by mAChR activation persists much longer and seems
to affect a significantly greater portion of the chromaffin cell
membrane. Based on estimates of the Ca2+
dependence of exocytosis from bovine chromaffin cells (Knight and
Baker, 1982; Augustine and Neher, 1992b) indicating that secretion can
be initiated at [Ca2+]i
lower than 1 µM, our results are consistent
with previous reports implicating mAChRs in eliciting secretion in rat
chromaffin cells (Wakade and Wakade, 1983; Malhotra et al., 1988
