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Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.
protein kinase C; channel phosphorylation; vascular smooth muscle; protein phosphatase
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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L-TYPE CALCIUM CHANNELS form a major pathway for Ca2+ entry into vascular smooth muscle (VSM) cells (26). These channels are modulated by distinct vascular agonists known to activate different second messenger systems, including the signaling cascade that activates protein kinase C (PKC) (2, 21, 24). The effect of PKC activation on functional properties of Ca2+ channels from smooth muscle cells has been investigated extensively but inconclusively (24, 35). Several lines of evidence have been presented indicating that PKC activators stimulate L-type Ca2+ currents in VSM cells obtained from different vascular territories (2). On the other hand, it has also been shown that phorbol ester activation of PKC was ineffective in modulating channel activity in smooth muscle cells from guinea pig basilar artery (25) and in the A7r5 cell line isolated from embryonic rat aorta (18, 19, 23, 33). When potentiation of Ca2+ currents by phorbol esters was previously observed in A7r5 cells, it was modest (<30%) (8), and in some cases activation was followed by inhibition after prolonged exposure (40).
The working hypothesis of the present work is that the different responses of Ca2+ channels in A7r5 cells to phorbol esters can be explained by different levels of resting phosphorylation, probably mediated by PKC, of the channel itself or of regulatory proteins. This hypothesis is consistent with three lines of indirect evidence previously reported in the literature: 1) A7r5 cells maintain a resting dihydropyridine-sensitive 45Ca2+ entry that is partially inhibited by staurosporine, a potent but nonspecific inhibitor of PKC (17); 2) A7r5 cells show high levels of PKC activity in membrane fractions in resting conditions (17); and 3) a positive correlation exists in A7r5 cells, under nonstimulated conditions, between L-type Ca2+ channel activity and PKC activity; both activities are upregulated by dexamethasone (18, 28).
The purpose of this study was to investigate whether L-type Ca2+ channels in A7r5 cells under resting conditions, in the absence of vascular agonists and with minimal serum stimulation, are phosphorylated by PKC and to characterize the kinetic mechanism underlying PKC modulation. Because phorbol esters did not affect Ca2+ channel activity in our experimental conditions, we speculated that these channels, or regulatory proteins associated with these channels, were already maximally phosphorylated. Therefore, the assumption was that, in resting conditions, the rates of protein phosphorylation far exceed the rates of protein dephosphorylation. This hypothesis suggested two possible scenarios that could be experimentally tested: 1) that Ca2+ channel activity should be insensitive to inhibitors of protein phosphatase and 2) that channel activity should be inhibited or decreased in the presence of PKC inhibitors through a mechanism that involves the activity of endogenous protein phosphatases.
Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by resting PKC activity, we observed that Ca2+ currents were not modulated by the phorbol ester phorbol 12,13-dibutyrate (PDBu) or by the protein phosphatase inhibitor okadaic acid. However, L-type Ca2+ currents were inhibited by PKC inhibitors, staurosporine and chelerythrine, through a mechanism that involves protein dephosphorylation. Inhibition of channel activity was due to a decrease in channel availability and long open events.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cell preparation. A7r5 cells were obtained from the American Type Culture Collection (Rockville, MD) and grown as described by Marks et al. (23). Briefly, cells were grown in DMEM containing 50 U/ml penicillin, 50 U/ml streptomycin, and 10% iron-supplemented calf serum. After cells reached confluence, the serum concentration was reduced to 0.5% for 1 wk. The day before the experiment, cell layers were dispersed with trypsin, resuspended in DMEM supplemented with 0.5% fetal calf serum, and plated on 4 × 4-mm Aclar films (Pro Plastics). Cytochalasin D was added to a final concentration of 0.14 µg/ml to maintain cells with a rounded morphology (23). This treatment was shown in different cell types not to affect PKC activity, PKC activation by phorbol esters, or cellular redistribution of the enzyme (3, 7, 39).
Experiments were carried out 1 day after plating. An Aclar film with cells attached to the surface was transferred into a 100-µl chamber perfused continuously at a rate of 25 µl/s with a solution (seal solution) containing (in mM) 150 NaCl, 1 CaCl2, and 2.5 NaHEPES (pH 7.35). Single cells separated from others in the field were chosen.Electrophysiology. Whole cell and single-channel currents were studied using an Axopatch-1B patch amplifier. Voltage commands were given and data were obtained using a microcomputer and a Labmaster analog-to-digital converter. pCLAMP software was used to acquire the experimental data. Electrodes were pulled from borosilicate glass (World Precision Instruments, New Haven, CT) and coated with silicone rubber.
Whole cell currents were measured with the fast (classical) (11) or the perforated patch whole cell configurations of the patch-clamp technique. Amphotericin B was used for the latter technique (32). The pipette solution used in the fast whole cell configuration contained (in mM) 20 CsCl, 100 cesium glutamate, 0.5 CaCl2, 5 MgCl2, 0.3 Na2GTP, 5 Na2ATP, 12 EGTA, and 10 HEPES (pH 7.2). The extracellular solution used in those experiments contained (in mM) 5 BaCl2, 138 NaCl, 10 HEPES, and 20 glucose (pH 7.35). The pipette solution for the perforated patch whole cell configuration contained 75 CsSO4, 55 CsCl, 5 CaCl2, and 10 HEPES (pH 7.35). The extracellular solution used in those experiments contained (in mM) either 5 BaCl2 or 5 CaCl2, 150 NaCl, 5 KCl, 10 HEPES, and 20 glucose (pH 7.35). Patch pipettes had resistances of 2-5 M
for classical whole cell and 1-2 M for perforated patch experiments. The access resistances were 2.9 ± 0.8 M
(n = 33) in classical whole cell
experiments and 5.9 ± 2.1 M (n = 20) in perforated patch experiments. Whole cell currents were
filtered at 1 kHz and sampled at 5 kHz. Currents were recorded within
the first 3 min after establishment of the whole cell configuration or
after a stable capacitive transient was reached in the perforated patch-clamp configuration. The effect of the intracellular dialysis of
okadaic acid, or vehicle, and the effects of externally applied PKC
activators and inhibitors were followed using voltage ramps from
90 to +90 mV, from a holding potential of 40 mV. Changes in whole cell Ca2+ currents were
evaluated using 60-ms step pulses to different membrane potentials.
Linear leakage and capacitive currents were subtracted using scaled
currents elicited by 2.5-mV hyperpolarizing pulses (P/4 protocol).
Single-channel currents were recorded using the cell-attached
configuration of the patch-clamp technique. The electrode solution contained (in mM) 110 BaCl2 and 10 N-methyl-D-glucamine-HEPES (pH
7.35). Electrodes had series resistances of ~3-5
M. Currents were filtered at 2 kHz and sampled at 10 kHz. Once the
cell-attached configuration of the patch-clamp technique was obtained,
the perfusion solution was changed to one containing (in mM) 140 potassium glutamate, 10 K-HEPES, 2 K-EGTA, and 2 MgCl2 to maintain a membrane
potential near 0 mV (depolarizing solution). At the same time, the
membrane patch was held at 40 mV, and 150-ms pulses to +10 mV
were delivered every 7 s.
Our standard protocol consisted of 48 pulses (each recorded every 7 s)
that defined a 5.6-min time window. The first time window, which
accounts for the equilibration with the depolarizing solution, was
excluded from the analysis. Our control window represents the following
48 records. After this sequence, cells were then perfused with
identical solutions supplemented with 100-300 nM PDBu or
50-100 nM staurosporine or 2-10 µM chelerythrine. The effects of these agents were evaluated by averaging channel activity from sweeps within the second identical time window after 5.6 min. For
controls, the depolarizing solution, alone or with the vehicle (0.05%
DMSO, vol/vol), was used. Experiments were carried out at room
temperature (~22°C).
Single-channel recordings were analyzed after subtraction of null
sweeps. The null sweeps were previously smoothed by fitting several
exponential functions and a constant component. The product (NPo) of
channel number (N) and channel open
probability
(Po) during the
pulse was calculated by dividing the number of samples in which channel
activity was above the 50% threshold by the total number of samples
(measured from 1 ms after the beginning of the pulse to the end). To
characterize the open time distribution, open events were detected
using the half-amplitude threshold criterion in current records,
interpolated with eight points between every two sample points (5).
During the analysis, currents were filtered at 1 kHz using a Gaussian
filter. The maximum likelihood method was used to fit the dwell time
distributions to the sum of several exponential functions, setting a
minimal value of 0.4 ms, which is equal to twice the system response
dead time (36). We used the likelihood ratio test to find the number of
exponential components required to fit a dwell time distribution.
Typically, the number of exponential functions was increased until the
fitted distributions were statistically insignificant
(P > 0.05) (15).
Chemicals and drugs. PDBu, staurosporine, and chelerythrine were from Sigma (St. Louis, MO) and Calbiochem (La Jolla, CA). Okadaic acid was from Calbiochem. DMSO was from Fisher.
Statistics. Values are shown as means ± SD except in Figs. 1B and 5, where error bars indicate SE. Mean values were compared using the t-test statistics from SigmaPlot. P values >0.05 were considered statistically insignificant.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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PDBu, an activator of PKC, does not affect L-type Ca2+ currents. The first step in the characterization of modulation of L-type Ca2+ channels by PKC was to investigate the effect of phorbol esters on whole cell currents. Results from other laboratories showed that acute exposure to phorbol esters produces no effect (23, 33) or a small initial increase of Ca2+ currents, sometimes followed by inhibition (8, 40). One possible explanation for the lack of effect of phorbol esters in whole cell experiments is that intracellular components necessary for PKC activation are dialyzed into the pipette after breaking the cell membrane (6). To investigate this possibility, we characterized the effect of PDBu in cell-attached patches, an experimental condition that preserves the intracellular milieu and allows longer recording times.
Figure 1A shows the current traces from one single-channel experiment in which the effect of 100 nM PDBu was investigated. The records from control, first, and third windows in PDBu are depicted sequentially to emphasize the lack of effect of PDBu. Figure 1B summarizes the results from seven experiments using 100-300 nM PDBu; the averaged NPo value of sweeps elicited each 7 s is plotted as a function of time. The lack of effect of PDBu on channel gating is supported by the occurrence of similar NPo values during the control window (4.5 ± 3.4%) and the second window in the presence of PDBu (2.6 ± 3.3%) ( P = not significant, paired t-test). In agreement with the single-channel experiments, no modulation of whole cell currents carried by 5 mM Ba2+ was observed in four experiments in the presence of 300 nM PDBu (not shown).
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Okadaic acid does not affect, whereas staurosporine decreases, L-type Ca2+ currents. One possible explanation for the lack of effect of phorbol esters on Ca2+ currents is that channels were already maximally activated by PKC under resting conditions. As indicated in the introduction, this possibility implies that 1) Ca2+ channels should also be insensitive to protein phosphatase inhibitors and 2) channels should be modulated by kinase inhibitors, provided resting protein phosphatase activity is present.
The first possibility, that Ca2+ channels are insensitive to protein phosphatase inhibitors, was confirmed using intracellular dialysis of 500 nM okadaic acid. This concentration is at least 20 times larger than the IC50 values of protein phosphatase (PP)-1 (20 nM) and PP-2A (0.2 nM) activities and similar to the IC50 value for PP-2B inhibition (500 nM) (4). Figure 2 shows that the current-voltage relationships after 10 min of intracellular dialysis with 500 nM okadaic acid (n = 6) and DMSO (n = 11) were similar. The lack of effect of okadaic acid is emphasized when the frequency distributions of the current densities at +20 mV are compared in Fig. 2, inset.
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40 to +20 mV after exposure to the vehicle, 100 nM staurosporine (n = 4), and 500 nM
staurosporine (n = 4) at the time
indicated by the arrow. Examples of current records are depicted in
Fig. 3C. To compare experiments with
different current densities, currents were normalized to the control
current density before exposure to the inhibitor. Staurosporine, but
not the vehicle, decreased Ca2+
currents in a concentration-dependent manner. This effect was not
reversible even after 10 min of washout (not shown). As expected for a
mechanism that involves protein dephosphorylation, the effect of 100 nM
staurosporine was prevented by okadaic acid, consistent with an
effective inhibition of protein phosphatases by the dialysis of okadaic
acid. In two experiments, we also confirmed that intracellular dialysis
of okadaic acid prevents the inhibitory effect of 500 nM staurosporine
(not shown).
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Kinetic mechanisms underlying the staurosporine effect. To investigate the gating mechanisms involved in Ca2+ current decrease due to PKC inhibitors, single-channel experiments were performed using the cell-attached configuration of the patch-clamp technique in the presence of 110 mM Ba2+ as current carrier. Figure 5A shows the average time course from 11 experiments in which the effect of 50-100 nM staurosporine on single-channel recordings was investigated. Staurosporine decreased channel activity after 5.6 min in 8 of 11 experiments. NPo values in the control (2.4 ± 2.7%) and during the second window in the presence of staurosporine (0.4 ± 0.4%) (Fig. 5A) were statistically different (P < 0.03, paired t-test). The staurosporine effect was not reversible (not shown). Figure 5B shows the average NPo value from 10 control experiments in which the effects of the depolarizing solution and the vehicle were investigated. Because no difference was observed between the two groups, we pooled all the data to show that the vehicle and the prolonged exposure to the high-K+, low-Ca2+ solution do not affect channel gating. The NPo values in the control window and during the second window were 2.4 ± 2.6 and 1.8 ± 2.1%, respectively ( P = not significant, paired t-test).
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(1) |
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1 = 0.3 ms, 2 = 0.7 ms, and
3 = 7.0 ms and fractions of
open events a1 = 0.812, a2 = 0.184, and
a3 = 0.004. The open time
distribution in the presence of staurosporine was fitted to two
exponential functions with 1 = 0.2 ms and 2 = 0.7 ms and
a1 = 0.827 and
a2 = 0.173. The absence of long
open events in the presence of staurosporine is interesting because it
is consistent with other reports showing that in VSM cells PKC
increases the appearance of long openings (34). Alternatively, this
effect could be due to a decreased likelihood of observing rare long
openings because of small sampling. We used the
G-test of independence with the
Williams correction (37) to investigate whether the proportion of long
open events differs in control and treated patches. The distribution of
G can be approximated by the
2 distribution with one degree
of freedom. Here we assumed that openings before and after
staurosporine had the same open time distribution, characterized by
short events, including openings from the two shorter exponential
components, and long open events [null hypothesis
(H0)]. From the fit of the
open time distribution, we estimated that, in control, 24 of 5,461 openings belonged to the population of long open events. On the other
hand, there was not a single long open event among the 726 estimated
openings during the second window in staurosporine. The
calculated G value was 5.64, and the
associated probability of occurrence of this arrangement under
H0 was 0.01 < P < 0.025, consistent with a direct inhibitory effect of staurosporine on long open events.
The effect of staurosporine on the latencies to first opening was
measured to evaluate the role of phosphorylation on the activation
kinetics of the channel. Figure 7C
shows that the cumulative first latency distributions in controls and
in the presence of staurosporine were similar, suggesting that
phosphorylation does not modulate the dwell time in the closed
transitions within the activation pathway of the channel. Moreover,
staurosporine did not affect the single-channel current amplitude
measured at +10 mV. This is shown in Fig.
7D, where the single-channel current was plotted as a function of the open duration of events recorded during the control and the second window in the presence of
staurosporine. In three cell-attached patches, 2-10 µM
chelerythrine also inhibited Ca2+
channel activity by decreasing the number of active sweeps (not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The experiments presented provide evidence that resting phosphorylation by PKC controls the gating kinetics of L-type Ca2+ channels in A7r5 cells in the absence of vascular agonists. Our experiments are consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under resting conditions, since 1) the phorbol ester PDBu was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases through intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid, suggesting the presence of a dephosphorylative process affecting the Ca2+ channel or regulatory proteins.
The lack of effect of PDBu under our experimental conditions is
consistent with previous reports in A7r5 cells and smooth muscle cells
from guinea pig artery (23, 25, 33). However, our findings are at
variance with studies in which phorbol esters potentiated
and/or inhibited Ca2+
channels in smooth muscle cells from human umbilical vein in a
dose-dependent manner (34). We suggest that this discrepancy may result
from the fact that Ca2+ channels,
or necessary regulatory proteins in A7r5 cells, are already
phosphorylated by PKC under resting conditions. Consistent with this
possibility, the protein kinase inhibitor H-7 has minimum effect on
Ca2+ channel activity in human
umbilical smooth muscle cells (34). Thus different preexisting levels
of kinase activity and phosphorylation would determine the type of
response of Ca2+ channels to PKC
activators. This condition may be similar to that observed in oocytes
and Chinese hamster ovary cells expressing the
1-subunit of the cardiac
Ca2+ channel when the modulation
of Ca2+ currents by protein kinase
A was investigated (30, 31). The increased resting channel
phosphorylation present in A7r5 cells may explain the high current
density showed by these cells compared with other VSM preparations
(23).
We have previously shown that treatment of A7r5 cells with
dexamethasone increases L-type
Ca2+ current density. However,
this effect was strongly dependent on the basal channel activity, as
only batches of cells showing low current density levels increased
activity upon treatment with dexamethasone (28). Interestingly,
dexamethasone does not affect the expression of the
Ca2+ channel
1-subunit in aortic cells (38)
but increases the content of PKC (18) and the activity of PKC in
both membrane and cytosolic fractions (17). Thus our results are
consistent with the possibility that different batches of cells
express, under nonstimulating conditions, different levels of PKC
activity and channel phosphorylation. This is probably the main reason
for the different results reported in the literature on the effect of
phorbol esters on Ca2+ currents in
A7r5 cells (8, 9, 23, 33).
The experiments presented provide evidence that the major effect of PKC is to control channel availability. This was directly assessed by measuring a decrease in the number of active sweeps after exposure to PKC inhibitors (Fig. 6). The possibility that the decrease in NPo was due to a major change in the Po was excluded because several kinetic mechanisms underlying this parameter were not affected. 1) The first latency distribution was similar before and after exposure to PKC inhibitors. This suggests that these compounds did not modulate the voltage-dependent and -independent mechanisms underlying the closed-closed transitions in the activation pathway. 2) The open time distribution in the presence of the inhibitors was comparable to that of control except for the absence of very rare long open events that are part of a distinct modal gating behavior (Fig. 7, A and B). 3) PKC inhibitors decrease whole cell currents with no effect on the shape of the current-voltage relationship (Figs. 3 and 4), suggesting that the voltage dependence of activation and the single-channel conductance were not affected. Currently, we cannot distinguish whether residual channel activity in the presence of PKC inhibitors results from dephosphorylated channels, showing a finite low probability of being available, or from the activity of channels phosphorylated by a lower, or additional, kinase activity.
The experiments also show that resting phosphorylation by PKC promotes
the presence of long open events, raising the issue of whether the
gating mechanisms underlying the long open events and
Pavail depend on
phosphorylation of multiple sites or, alternatively, whether
phosphorylation of a single site modulates both fast and slow gating
kinetics. Evidence for independent phosphorylation sites controlling
fast and slow gating of Ca2+
channels has been obtained in myocardial cells (29, 41), where
phosphatase inhibitors increased
Ca2+ channel availability as well
as the fraction of long open events. On the basis of the different dose
responses required for these two effects, it was suggested that
independent phosphorylation events modulate both kinetic behaviors.
Additional evidence for independent phosphorylation sites in cardiac
Ca2+ channels derives from the
kinetic analysis of Ca2+ currents
stimulated by 
-adrenergic agonists (13, 14) and the observed shift
between gating modes (42). At variance with the effect of -agonists
on myocardial cells, phorbol esters produce a biphasic effect on
channel gating, showing first an increase and then a decrease in
channel availability, with no effect on open time duration (20).
Studies using protein phosphatase inhibitors in human umbilical smooth
muscle cells also show that channel availability and long open events
are controlled by different phosphorylation sites (10, 34).
Taken together, these results suggest that under resting conditions, L-type Ca2+ channels in A7r5 cells require full PKC activity to open upon depolarization. This information is the key to understanding the consequence of Ca2+ channel modulation (and Ca2+ entry) in VSM cells by agonists that activate the PKC cascade. Different resting levels of channel phosphorylation will determine different resting cytosolic Ca2+ concentrations (17) and different fractional increases of Ca2+ entry upon depolarization.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-41618 and American Heart Association (Northeast Ohio Affiliate) postdoctoral fellowships to C. A. Obejero-Paz and M. Auslender.
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FOOTNOTES |
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Preliminary results were previously presented in abstract form (27).
Present address of M. Auslender: Div. of Pediatric Cardiology, School of Medicine, New York University, New York, NY 10016.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: C. A. Obejero-Paz, Dept. of Physiology and Biophysics, School of Medicine, 2109 Abington Rd., Cleveland, OH 44106.
Received 18 February 1998; accepted in final form 8 May 1998.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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;
n = 6) and DMSO (
;
n = 11) on whole cell currents carried
by 5 mM Ba2+. Currents were
elicited by voltage steps from a holding potential of 
, DMSO used in
intracellular dialysis and DMSO added at arrow; 
, DMSO in dialysis and 500 nM staurosporine added at arrow. B:
current-voltage relationship from 4 experiments before (
