Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, Vermont 05405
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ABSTRACT |
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Scornik, Fabiana S.,
Laura A. Merriam, and
Rodney L. Parsons.
Number of KCa Channels Underlying Spontaneous
Miniature Outward Currents (SMOCs) in Mudpuppy Cardiac Neurons.
J. Neurophysiol. 85: 54-60, 2001.
Spontaneous miniature outward currents (SMOCs) in parasympathetic
neurons from mudpuppy cardiac ganglia are caused by activation of TEA-
and iberiotoxin-sensitive, Ca2+-dependent K+
(BK) channels. Previously we reported that SMOCs are activated by
Ca2+-induced Ca2+ release (CICR) from caffeine-
and ryanodine-sensitive intracellular Ca2+ stores. In the
present study, we analyzed the single channel currents that contribute
to SMOC generation in mudpuppy cardiac neurons. The slope conductance
of BK channels, determined from the I-V relationship of
single-channel currents recorded with cell-attached patches in
physiological K+ concentrations, was 84 pS. The evidence
supporting the identity of this channel as the channel involved in SMOC
generation was its sensitivity to internal Ca2+, external
TEA, and caffeine. In cell-attached patch recordings, 166 µM TEA
applied in the pipette reduced single-channel current amplitude by
32%, and bath-applied caffeine increased BK channel activity. The
ratio between the averaged SMOC amplitude and the single-channel
current amplitude was used to estimate the average number of channels
involved in SMOC generation. The estimated number of channels involved
in generation of an averaged SMOC ranged from 18 to 23 channels. We
also determined that the Po of the BK channels at the
peak of a SMOC remains constant at voltages more positive than 20 mV,
suggesting that the transient rise in intracellular Ca2+
from ryanodine-sensitive intracellular stores in the vicinity of the BK
channel reached concentrations most likely exceeding 40 µM.
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INTRODUCTION |
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Parasympathetic cardiac neurons,
like other autonomic neurons, express a number of different voltage-
and ligand-gated potassium channels in their plasma membranes that are
critical determinants of membrane potential and neuronal excitability
(Adams and Harper 1995; Brown 1988
;
Rudy 1988
). Among these different potassium conductances, the Ca2+- and voltage-activated
potassium (KCa) channels have been shown particularly to play a key role in determining resting membrane potential, action potential repolarization, and post action potential hyperpolarizations (Kaczorowski et al. 1996
; Rudy
1988
; Sah 1996
). Activation of
KCa channels is very dynamic because it is gated by elevation of the Ca2+ concentration at the
intracellular side of the channel. Very localized elevations of
Ca2+, which do not significantly elevate global
intracellular Ca2+, can activate
KCa channels. Thus physiological activation of KCa channels may be coupled to highly localized
changes of cell Ca2+.
Cytoplasmic Ca2+ concentration is generally
maintained at very low levels. However, Ca2+
levels can be elevated locally for a brief period following
Ca2+ influx or following
Ca2+ release from internal stores. Neurons, like
most cell types, exhibit IP3- or ryanodine- and
caffeine-sensitive Ca2+ pools that are involved
in the release of Ca2+ from intracellular stores
(Berridge 1993; Henzi and MacDermott 1992
; Kuba 1994
; Verkhratsky and Shmigol
1996
). It has been hypothesized for neurons that release of
Ca2+ from the ryanodine-sensitive stores may
amplify and prolong intracellular Ca2+ signaling
by the process of Ca2+-induced
Ca2+ release (CICR) (Hua et al.
1993
).
Brief, highly localized release of Ca2+ from
internal stores has been demonstrated in cardiac, skeletal, and smooth
muscle cells (Cheng et al. 1993; Imaizumi et al.
1999
; Klein et al. 1996
; Nelson et al.
1995
). In smooth muscle cells, these increases of
Ca2+ activate large-conductance
KCa (BK) channels in the plasma membrane to cause
transient outward currents (Benham and Bolton 1986
;
Nelson et al. 1995
).
Transient hyperpolarizations or outward currents have also been
recorded from neurons (Adams et al. 1985;
Fletcher and Chiappinelli 1992
; Hartzell et al.
1977
; Marrion and Adams 1992
; Mathers and Barker 1981
, 1984
; Merriam et al. 1999
;
Munakata and Akaike 1993
; Satin and Adams
1987
). Satin and Adams (1987)
showed that
activation of BK channels produced spontaneous miniature outward
currents (SMOCs), which were responsible for the transient
hyperpolarizations initially noted by Hartzell et al.
(1977)
. Recently we examined, in more detail, the
characteristics of SMOCs recorded from dissociated mudpuppy
parasympathetic cardiac neurons. We showed that SMOCs are reduced by
low concentrations of TEA and eliminated by iberiotoxin, a specific
blocker of BK channels (Merriam et al. 1999
). We also showed that SMOCs are activated by a CICR-type mechanism that required
the release of Ca2+ from caffeine- and
ryanodine-sensitive stores.
In previous reports, the estimated number of BK channels needed to
activate a SMOC have ranged widely, from a few to many thousands
(Fletcher and Chiappinelli 1992; Satin and Adams
1987
). The present study was done to establish the number of BK
channels that are activated simultaneously to initiate SMOCs in
mudpuppy neurons and to estimate the Ca2+
concentration needed for this activation. To answer these questions, we
used cell-attached patch-clamp and excised-patch recording techniques
to establish the properties of BK channels present in the plasma
membrane of mudpuppy parasympathetic cardiac neurons. We then compared,
over a range of voltages, BK channel current and averaged SMOC
amplitudes to establish the number of individual channels involved in
SMOC generation. Our results indicated that approximately 20 (range
18-23) BK channels participate in the generation of an averaged SMOC,
and furthermore, that the open probability (Po) of the BK
channels at the peak of a SMOC is near saturation for voltages more
positive than
20 mV. This latter observation, combined with direct
measurements of BK channel Ca2+ sensitivity,
indicated that SMOCs are activated by a transient rise of intracellular
Ca2+ in the vicinity of the BK channels, which
reaches concentrations locally equal to or greater than 40 µM.
A preliminary description of this work has been presented previously
(Scornik et al. 1999).
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METHODS |
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All experiments were performed on parasympathetic neurons
dissociated from mudpuppy (Necturus maculosus) cardiac
ganglia. Mudpuppies were killed by rapid decapitation, following
procedures approved by the University of Vermont Institutional Animal
Care and Use Committee. The method of dissociation used a combination of collagenase, type I (Sigma Chemical, St. Louis, MO) and neutral protease (Sigma) following methods described previously (Merriam and Parsons 1995). All experiments were completed at room
temperature (21-22°C).
Electrophysiological methods
Whole cell current recordings were made using the
perforated-patch configuration of the whole cell patch-clamp technique
(Horn and Marty 1988) and were controlled using an
Axopatch-1C/Labmaster DMA TL-1-125/pClamp 6.0.3 acquisition system
(Axon Instruments, Foster City, CA). Currents were filtered at 2 kHz,
stored on tape using a PCM recorder (AR Vetter, Rebersburg, PA), and
then digitized (200 µS) for further analysis using the SCAN program
(Strathclyde Electrophysiology Software, John Dempster, University of
Strathclyde, Glasgow, Scotland). SMOCs were collected for at least 2 min at holding potentials ranging between
40 and +40 mV. Peak
amplitudes of individual SMOCs were determined using a
threshold-detection utility that was set to detect events that were
distinguishable from baseline noise. This threshold was established by
examining digitized files by eye and varied with each potential,
ranging from 6 pA at more hyperpolarized potentials to 15 pA at the
most depolarized potentials. Average SMOC amplitude was determined by
averaging at least 100 SMOCs at each voltage, except for voltages in
which the SMOC frequency was greatly decreased. As we reported previously (Merriam et al. 1999
), SMOC frequency is
voltage dependent, being lower at those voltages at which
Ca2+ influx through voltage-dependent
Ca2+ channels is reduced. For those voltages, a
minimum of 25 events were averaged to determine SMOC amplitude. Series
resistance compensation was used as described previously
(Merriam and Parsons 1995
), and the amount of
uncompensated series resistance was monitored throughout recordings.
Reported voltages were corrected for voltage-clamp error due to
uncompensated series resistance and for a junction potential of 10 mV
(JPCalc, Cell Microcontrols, Virginia Beach, VA).
Single-channel current recordings were made using either the
cell-attached or the inside-out excised patch variation of the patch-clamp technique (Hamill et al. 1981).
Voltage-clamp experiments were controlled with an Axopatch 200/Digidata
1200/pClamp 6.0.3 acquisition system (Axon Instruments). Currents were
filtered at 2 kHz, stored on tape using a PCM recorder (AR Vetter), and then digitized (100-400 µS) for further analysis using pClamp 6.0.3 or pClamp 5.6 acquisition software. Single-channel records were
analyzed for current amplitudes and open channel probabilities using
TRANSIT (written by Dr. A.M.J. Vandongen, Duke University, Durham, NC).
For single-channel current recordings, the patch membrane potential was
controlled by applying voltage to the external side of the membrane
patch. In whole cell perforated-patch recordings made in current-clamp
mode, the averaged resting membrane potential for dissociated mudpuppy
neurons recorded in normal physiological solution was 50.5 ± 1.5 mV (n = 9). Consequently, for the cell-attached patch recordings of single-channel activity,
50 mV was assumed to
approximate the resting membrane potential for cells when no potential
was applied to the pipette. The voltages reported for single-channel
currents obtained from cell-attached patches were adjusted to account
for the resting membrane potential.
Solutions for SMOC recordings
The bath solution for SMOC recordings contained 110 mM NaCl, 3.6 mM CaCl2, 2.5 mM KCl, 10 mM NaHEPES, 0.3 µM
tetrodotoxin (TTX), and 100 µM CdCl2, pH 7.3. In 4 of 11 cells, 100 nM apamin was included to block SK channels. The
presence of apamin did not affect SMOC characteristics (Merriam
et al. 1999). The pipette solution was (in mM) 80 K-aspartate,
40 KCl, 5 MgCl2, and 10 HEPES-KOH, pH 7.2. The
patch pipettes were backfilled with 0.2 mg/ml amphotericin B (Sigma).
Solutions for single-channel current recordings
The bath solution for the cell-attached patch recordings contained in (mM) 110 NaCl, 3.6 CaCl2, 2.5 KCl, and 10 NaHEPES, pH 7.3. The pipette solution was the same with the addition of 200 nM apamin, 0.3 µM TTX, and 1.4 mM MgCl2. The bath solution for the inside-out excised patch recordings contained (in mM) 120 KCl, 20 NaCl, 10 HEPES-KOH, 5 HEDTA, and 1 MgCl2 as well as varying Ca2+ concentrations (0.35, 1, 5.5, 10, 30, 40, or 100 µM), pH 7.2. The Ca2+ concentrations were measured with a Ca2+ electrode (WPI, Sarasota, FL).
Drugs
All drugs used in the present study were obtained from commercial sources as indicated. Tetraethylammonium (TEA), caffeine, apamin (Sigma) and tetrodotoxin (TTX) (Alomone Labs, Jerusalem, Israel) were prepared as aqueous stocks before use. Ionomycin (Sigma) was prepared as a DMSO stock (1:1500 dilution).
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RESULTS |
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Voltage dependence of SMOC and BK current amplitude
In the first series of experiments, we determined, using
perforated-patch whole cell recordings, the averaged peak current amplitude versus voltage (I-V) relationship for mudpuppy
SMOCs. In these experiments, 100 µM Cd2+ was
included in the bath solution. We had previously reported that
Cd2+ does not affect SMOC amplitude but decreases
the frequency of SMOCs, thus making it possible to analyze individual
events (Merriam et al. 1999). Figure
1A illustrates a typical
I-V relationship for SMOCs recorded from mudpuppy cardiac
neurons. Similar results were observed in 10 additional experiments,
shown in Fig. 4A.
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Single-channel currents were recorded with neurons bathed in the control physiological solution (see METHODS), without Cd2+ and with a similar solution inside the patch pipette. Single-channel currents were initially recorded from cell-attached patches without any applied voltage and then after negative voltages were applied to the pipette to depolarize the membrane inside the patch. The I-V relationship was determined for a large-conductance channel recorded in five individual experiments. The average conductance of this channel determined from the slope of the I-V relationship was 84 ± 4 (SE) pS. Results from one of these experiments are shown in Fig. 1B.
To establish that this large-conductance channel was a BK channel, additional single-channel current recordings were made in which we tested the sensitivity of this channel to blockade by TEA. TEA sensitivity was studied by back filling one-third of the recording pipette with 500 µM TEA. Patches were obtained, and TEA was allowed to equilibrate within the pipette with the final TEA concentration at the membrane reaching approximately 166 µM. For these experiments, the Ca2+ ionophore, ionomycin, was also included in the pipette solution to increase BK channel activity so that block by TEA would be more evident. The pipette solution was added in three layers (1st pipette solution, then pipette solution with ionomycin, and finally pipette solution with ionomycin and TEA). Immediately after a seal was formed, BK channel activity was low, but then over time, channel activity increased, an indication that ionomycin had reached the membrane and Ca2+ influx through the patch membrane had started. Although channel activation increased, the peak single-channel current amplitude was not affected by ionomycin (data not shown). The onset of the increase in channel activity was a good indication of the time needed for the ionomycin containing solution to diffuse to the membrane and provided a means to estimate when TEA would reach the membrane. Currents recorded during ionomycin exposure, but before TEA had reached the membrane, and then after TEA block, are shown in Fig. 2B. Once the TEA-induced decrease in current amplitude was evident, current recordings were made at different membrane potentials. From the I-V relationship presented in Fig. 2A, we estimated the slope conductance of the channel blocked by TEA in this example to be 32 pS. In three experiments, the average decrease in single-channel current amplitude by TEA was 32 ± 8%.
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Blockade by TEA provided evidence that the 84-pS channel was a BK
channel. As a further test, we determined whether channel activity
could be increased by caffeine-induced Ca2+
release from caffeine-sensitive internal stores. Activation of BK
channels by caffeine in rat cardiac neurons was previously reported by
Smith and Adams (1999). In our earlier study, we
demonstrated that SMOC frequency was increased by caffeine-induced
Ca2+ release (Merriam et al.
1999
). We reasoned that if the 84-pS channels were involved in
SMOC generation, then their activity should be increased by a
caffeine-induced elevation of intracellular Ca2+.
This was tested in experiments in which caffeine was added to the bath
solution at a final concentration of 3 mM and the patch membrane was
depolarized. In three experiments, channel activity increased following
the addition of caffeine to the bath (Fig. 2C); thus
confirming that, as previously observed for SMOCs, the single-channel
activity is affected by Ca2+ release from
caffeine-sensitive stores.
Ca2+ activation curve for mudpuppy BK channels
Although the increase in channel activity induced by ionomycin, as
well as by caffeine, suggested that the 84-pS channel was Ca2+ sensitive, documentation of direct
Ca2+ activation of the channel was required. In
addition, to estimate the concentration of Ca2+
that activates SMOCs, it was necessary to know the open probability (Po) versus Ca2+ relationship for BK
channels in the mudpuppy neurons. Therefore additional experiments were
done to establish the Ca2+ sensitivity of these
BK channels. To accomplish this, single-channel currents were recorded
from excised patches in the inside-out recording mode. The pipette
solution was the normal physiological solution (see
METHODS), and the bath solution, which faced the cytoplasmic side of the channel, contained 120 mM KCl, to approximate the intracellular K+ concentration. The
Ca2+ concentration of the bathing solution was
adjusted to different values using a Ca2+
chelator; each Ca2+ concentration was measured
with a Ca2+ electrode. In these experiments,
seals were obtained with the cells bathed in control solution
containing 3.6 mM CaCl2 and 2.5 mM KCl. Following
seal formation, the presence of channel activity was determined by
transiently depolarizing the patched membrane. If channel activity was
evident, then the patch was excised and the control bath solution
replaced by the 120 mM KCl solution with adjusted
Ca2+. Single-channel current recordings were made
in the presence of different Ca2+ concentrations
with channel activity recorded in each condition for a minimum time of
5 min at 0 mV. During this time, there was no evidence, at any of the
Ca2+ concentrations, of channel inactivation as
had been previously reported for BK channels in chromaffin cells
(Solaro and Lingle 1992). The Po of the
channel was calculated for each Ca2+ level and
then plotted as a function of the different Ca2+
concentrations (Fig. 3A). The
open probability-Ca2+ relationship was fitted
with a Hill function giving a Ca50 of 3.5 µM
and a Hill coefficient of 2.7. The maximum Po obtained in
these conditions was 0.83, and it was reached at 40 µM
Ca2+. In Fig. 3A, data were included
from experiments in which only one channel was detected
(n = 3). However, assuming that the number of current
levels observed represented the number of channels in the patch and
that Po was equal for all the channels, similar results were
obtained from three additional experiments in which two or three levels
of channel activity were observed (data not shown).
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It is well established that membrane voltage influences BK channel
Ca2+ sensitivity. To confirm this for the
mudpuppy neurons, we recorded channel activity at different membrane
potentials. The single-channel current records presented in Fig.
3B were obtained with the membrane patch held either at 40
or 0 mV and exposed to various Ca2+
concentrations. In this example, it is observed that for the same
Ca2+ concentration, channel activity was lower at
40 than at 0 mV. However, when the Ca2+
concentration was raised from 5.5 to 40 µM, the difference was diminished. Table 1 shows the averaged
values of Po for three cells recorded at the same voltages
and calcium concentrations as Fig. 3B. These observations
are consistent with previous conclusions that the voltage sensitivity
of BK channels shifts with increasing internal
Ca2+ concentration (Adams et al.
1982
; Barrett et al. 1982
).
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Number of BK channels comprising an averaged SMOC
Knowing the I-V relationship for both averaged SMOCs
and BK channel currents allowed a calculation of the number of channels that are activated to create an averaged SMOC at different voltages. This estimation of the number of single channels per averaged SMOC was
based on the fact that the peak SMOC amplitude, I, is determined by the relationship I = NPo(Vm
Veq) and the single-channel current
amplitude, i, is given by i =
(Vm
Veq),
where
is the single-channel conductance, N is the number
of channels, Po is the single channel open probability,
Vm is the membrane potential, and
Veq is the equilibrium potential for
K+. Thus at a given voltage, the ratio between
the whole cell current I and the single-channel current
i will give NPo at that voltage. Figure
4, A and B, shows
the I-V relationships for SMOCs and single-channel currents
obtained in different experiments. SMOCs were recorded at different
voltages in 11 cells, and the combined data are shown in Fig.
4A. In Fig. 4B, the single-channel current
amplitude versus voltage relationship was the averaged data for five
cell-attached experiments with the resting membrane potential assumed
to be
50 mV (see METHODS). At potentials close to the
equilibrium potential for K+, the single-channel
currents exhibited rectification (data not shown). Consequently, the
single-channel data analysis was confined to potentials equal to or
more positive than
50 mV, where the data could be adequately
described by a linear function. Each value from the SMOC I-V
relationship was divided by the corresponding value at the same voltage
in the linear fit of the I-V relationship for the single
channels. The values resulting from this ratio were taken as
NPo and are shown in Fig. 4C. The combined data points in Fig. 4C were fit by a Boltzmann relationship:
Po = 1/{1 + exp[(V
V1/2)/k]}, where
V is the membrane voltage,
V1/2 is the half activation voltage,
and k is the Boltzmann constant. Assuming a value for
k = 12, chosen from published values for BK channels
from neurons (Dopico et al. 1999
; Tseng-Crank et
al. 1994
), the V1/2 calculated
from the combined data was
47.8 mV.
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Two points were evident from inspection of Fig. 4C. First,
the calculated NPo for the averaged SMOCs at +37 mV, the
most depolarized potential, was 18, and second, the NPo did
not change appreciably over the voltage range 20 to +37 mV with
values ranging from 16 at
20 mV to 18 at +37 mV. The apparent
insensitivity to membrane voltage suggested that the rise in
Ca2+ concentration near the channel must approach
values that shift the voltage activation curve close to saturation in
the range of
20 to +37 mV, very likely exceeding 40 µM.
Next we generated a family of Po versus voltage curves by
substituting different hypothetical values for Po at +37 mV.
In Fig. 4D, three different Po curves were
constructed using the Boltzmann relation from Fig. 4C,
assuming maximum values for Po at +37 mV of 0.5 (· · ·), 0.8 (- - -), and 1 (). The number of channels available, N, at +37 mV, with Po equal
to 1, 0.8, or 0.5 would be 18, 23, or 36, respectively.
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DISCUSSION |
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In our previous study, we demonstrated that SMOCs in mudpuppy
parasympathetic cardiac neurons are activated by a CICR-type mechanism
mediated by Ca2+ release from ryanodine- and
caffeine-sensitive stores. The results of our prior work also strongly
suggested that the endoplasmic reticulum Ca2+
release channels must be very close to the BK channels in the plasma
membrane and that the Ca2+ concentration in the
confined space near these channels must be high (Merriam et al.
1999). The present study extended these observations to
determine the number of BK channels that would contribute to the
generation of SMOCs over a range of voltages and confirmed the
suggestion that the transient release of Ca2+
from internal stores must increase the local Ca2+
concentration to high levels. Two major conclusions can be drawn from
results obtained in the present study. First, the estimated average
number of BK channels involved in the generation of an averaged SMOC
ranged between 18 and 23. Second, the Ca2+
concentration in close proximity to the BK channels at the peak of a
SMOC must exceed 40 µM.
We have established the relationship between averaged SMOC current
amplitude and membrane potential (Figs. 1 and 4A). At any voltage, the peak amplitude of a SMOC should depend on three primary factors: the unitary conductance () of the channel, the
driving force for K+
(Vm - Veq) that increases as the membrane
potential becomes more positive, and the number of channels open. The
number of channels open at the peak of a SMOC is a function of both the
total number of channels (N) and the probability
(Po) that a channel will open. Dividing the averaged SMOC
amplitude by the mean single-channel current provided an estimate of
NPo over the voltage range
40 to +37 mV. At membrane
potentials greater than
20 mV, NPo values did not change
appreciably, ranging from 16 at
20 mV to 18 at +37 mV. If we assumed
a Po value between 0.5 and 1, the number of channels
activated at +37 mV would range between 18 and 36. However, an estimate
for Po of 0.5 at +37 mV was considered very unlikely. From
the Ca2+ activation curve (Fig. 3A), a
Po equal to 0.5 would occur when the
Ca2+ concentration in the vicinity of the BK
channels was only a few micromolar. At this concentration of
Ca2+, the reported value for the
V1/2 of BK channels in neurons is approximately 0 mV (Tseng-Crank et al. 1994
). The value
of V1/2 reported in this paper is
48
mV. Thus the voltage dependence of BK channel activation is shifted to
the left along the voltage axis, suggesting that the
Ca2+ concentration must be much greater than a
few micromolar. Therefore we concluded that the Po would be
more in the range of 0.8 to 1 at +37 mV, and the number of BK channels
contributing to the generation of an averaged SMOC would vary between
18 and 23. V1/2 values reported for BK
channels from neurons at 400 µM Ca2+ ranged
from
38 to
50 mV (Tseng-Crank et al. 1994
). In
addition, we determined a V1/2 =
47
mV for mudpuppy BK channels exposed to 300 µM
Ca2+ (Scornik and Parsons, unpublished
observations). Thus the V1/2 value of
48 mV obtained from the analysis of BK channels underlying SMOCs
strongly suggested that the Ca2+ concentration at
the peak of a SMOC must be higher than 40 µM.
Direct measurements of the Ca2+ sensitivity support this conclusion. Data obtained from inside-out patches showed that the BK channels exhibited a Ca2+-activation relationship with a Ca50 of 3.5 µM. The maximum Po at 0 mV was 0.83 and occurred with 40 µM Ca2+. Consequently, we suggest that at the peak of the SMOC, the local Ca2+ concentration must be at least 40 µM, perhaps reaching concentrations considerably greater.
SMOCs are very brief, exhibiting a half decay time of 7.6 ± 0.3 ms between 10 and 0 mV (n = 11). It has been
suggested that the decay phase of SMOCs is determined by BK channel
closing (Merriam et al. 1999
; Satin and Adams
1987
). Kinetic analysis of single-channel currents from three
patches supported this idea. The mean open and closed times at 40 µM
Ca2+ and at 0 mV were 18.1 ± 4.6 and
1.68 ± 0.3 ms, respectively. In addition, we performed a burst
analysis for the channels measured at 5.5 µM
Ca2+ and at 0 mV. The results showed that even
when considering a critical closed time as short as 5 ms, the duration
of the burst (71.6 ± 8 ms, n = 3) greatly
exceeded the averaged SMOC 50% decay time at that voltage. Although a
more detailed analysis of the single-channel currents underlying SMOCs
is necessary to explain SMOC behavior, our data suggest that BK channel
closing kinetics, rather than bursting behavior, controls SMOC decay.
Thus the local Ca2+ concentration must decay very
rapidly, suggesting that very efficient mechanisms rapidly reduce the
Ca2+ concentration in the microdomain near the
cytoplasmic face of the BK channel.
From the Ca2+ activation curve shown in Fig. 3,
the single-channel Po in the presence of 3.5 µM
Ca2+ was about 0.4. Assuming the simplest model,
at 0 mV, the half-decay time of the SMOC would be the time for the
local Ca2+ concentration to change from more than
40 to 3.5 µM, a concentration at which approximately half of the
activated channels would have closed. Such a rapid removal of
Ca2+ indicates that Ca2+
homeostasis is very tightly regulated in these cells. Neurons are known
to have a number of very efficient Ca2+-buffering
systems (Sadoshima and Akaike 1991; Tillotson and
Gorman 1980
). The mechanism(s) responsible for removing
Ca2+ and terminating SMOCs has not been
identified. However, results presented in our previous study indicated
that Ca2+ uptake by the endoplasmic
reticulum Ca2+ATPase was not involved because
blockade of the Ca2+ATPase by cyclopiazonic acid
did not affect SMOC decay time or amplitude (Merriam et al.
1999
). Also, we cannot exclude the possibility that diffusion
is responsible for rapid removal of Ca2+
from the vicinity of the channel.
Recently in smooth muscle cells, fluorescence measurements of
transient, localized elevations of intracellular
Ca2+ (Ca2+ sparks) have
been correlated and recorded simultaneously with spontaneous transient
outward currents (STOCs) (Pérez et al. 1999;
ZhuGe et al. 1998
, 1999
). Similar measurements have not been completed to date in neurons. We have initiated experiments to
image "neuronal Ca2+ sparks" that might be
correlated with SMOCs. The Ca-sensitive dyes [Fluo-3, and Oregon green
488 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)] that have been employed in our laboratory to date should
be sensitive enough to detect a Ca2+ change from
resting levels to more than 40 µM. The limited success to date
reinforces our suggestion that the distance between the site of
Ca2+ release and the BK channels must be
extremely small, and furthermore that the transient elevation of
intracellular Ca2+ must be terminated very
rapidly. Previously, we noted that incubation with the rapid
Ca2+ chelator BAPTA eliminated SMOC activity.
However, even after SMOC activity had ceased, application of caffeine
could re-initiate SMOC activity in BAPTA-treated cells (Merriam
et al. 1999
), indicating that the Ca2+
release sites must be within 10-20 nm of the cytoplasmic surface of
the BK channels (Neher 1998
).
In conclusion, the data presented in this study suggest that the averaged number of BK channels involved in the generation of an averaged SMOC in mudpuppy cardiac neurons ranged from 18 to 23. Also our results indicate the concentration of Ca2+ in the vicinity of the BK channels at the peak of a SMOC must reach values more than 40 µM. The rise in Ca2+ that activates the averaged 18-23 channels comprising a SMOC must be efficiently and rapidly removed from the cytoplasmic face of the channel in order for the SMOC to exhibit its characteristic rapid decay.
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ACKNOWLEDGMENTS |
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The authors thank Dr. John Dempster for providing the SCAN program and Drs. Mark Nelson, Joseph Patlak, and Ariel Escobar for critical comments during preparation of the manuscript.
This work was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-23978 to R. L. Parsons and American Heart Association Grant 9820031T to F. S. Scornik.
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FOOTNOTES |
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Address for reprint requests: R. L. Parsons (E-mail: rparsons{at}zoo.uvm.edu).
Received 30 March 2000; accepted in final form 14 September 2000.
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