PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

C. A. Obejero-Paz, M. Auslender, and A. Scarpa

Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 MOmega for classical whole cell and 1-2 MOmega for perforated patch experiments. The access resistances were 2.9 ± 0.8 MOmega (n = 33) in classical whole cell experiments and 5.9 ± 2.1 MOmega (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 MOmega . 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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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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Fig. 1.   Phorbol 12,13-dibutyrate (PDBu) does not modulate L-type Ca2+ channel activity in cell-attached patches. A: single-channel records from 1 experiment using 100 nM PDBu to activate protein kinase C (PKC). Step pulses to +10 mV were delivered from a holding potential of -40 mV every 7 s. All records from control window and first and third windows after addition of PDBu are depicted. Single-channel openings are shown as downward deflections of baseline (patch 96425). B: values of product (NPo) of channel number (N) and channel open probability (Po) from 7 experiments in which effects of 100 nM (n = 6) and 300 nM (n = 1) PDBu were assessed. NPo values (means ± SE) from individual sweeps in different experiments were averaged at each time point and plotted as a function of time. Solid line, presence of PDBu. Dashed lines, control window and second window in presence of PDBu.

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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Fig. 2.   Effect of intracellular dialysis of okadaic acid (bullet ; n = 6) and DMSO (open circle ; n = 11) on whole cell currents carried by 5 mM Ba2+. Currents were elicited by voltage steps from a holding potential of -40 mV. Inset: distribution of current densities at +20 mV in both experimental conditions.

Staurosporine was used to test the second possibility that maximally phosphorylated channels should be inhibited by protein kinase inhibitors. Figure 3A shows the time course of the changes of whole cell currents elicited by step pulses from -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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Fig. 3.   A: effect of staurosporine (stau) on whole cell Ca2+ currents carried by 5 mM Ba2+. star , DMSO used in intracellular dialysis and DMSO added at arrow; open circle , DMSO in dialysis and 100 nM staurosporine added at arrow; bullet , okadaic acid in dialysis and 100 nM staurosporine added at arrow; triangle , DMSO in dialysis and 500 nM staurosporine added at arrow. B: current-voltage relationship from 4 experiments before (open circle ) and 10 min after (bullet ) exposure to 100 nM staurosporine. Currents were normalized to control current recorded at +20 mV. C: current traces representative of 4 distinct experimental conditions. First compound indicated for each trace was used in intracellular dialysis, and staurosporine (at concentrations indicated) or vehicle (DMSO) was added at time indicated by arrow in A. Numbers at end of each current trace indicate time (in min) elapsed from start of intracellular dialysis. * Control traces.

Figure 3B shows the effect of staurosporine on the current-voltage relationship from four experiments. To compare the voltage dependence of the currents inhibited by staurosporine, whole cell currents at different membrane potentials under control conditions and after treatment with staurosporine were normalized to the current elicited at +20 mV during the control. Both current-voltage relationships showed approximately the same shape, suggesting that PKC inhibition did not affect the reversal potential and voltage dependence of the Po.

Taken together, these results suggest that L-type Ca2+ channels in A7r5 cells are maximally phosphorylated under resting conditions. To confirm that this phosphorylation was due to resting PKC activity, a more specific PKC inhibitor, chelerythrine, was used. Chelerythrine inhibits PKC with an IC50 of 0.7 µM, a concentration at least two orders of magnitude smaller than that required to inhibit protein kinase A (0.17 mM), tyrosine protein kinase (0.1 mM), and Ca2+/calmodulin-dependent protein kinase (>0.1 mM) (12). To decrease Ca2+ current rundown, whole cell currents were recorded using the perforated patch-clamp configuration. Figure 4A shows the current-voltage relationship from two perforated patch-clamp experiments using 5 mM Ca2+ as current carrier before and 10 min after exposure to 2 µM chelerythrine. Figure 4A, inset, shows the currents recorded at +10 mV in the presence and absence of chelerythrine. Similar to the effect observed using the classical whole cell configuration, the decrease in current resulting from inhibition of PKC activity was approximately constant over the range of membrane potentials investigated. Figure 4B shows the time course of the current changes after the vehicle (2 experiments) or chelerythrine (3 experiments) was added at time 0. For comparison, we also show two of the four perforated patch-clamp experiments in which 100 nM staurosporine inhibited channel activity. Because the inhibitory effects of both PKC inhibitors on Ca2+ currents recorded in 5 mM Ba2+ or 5 mM Ca2+ were comparable, we pooled the experimental results together. To compare experiments with different current densities, currents were normalized to control before exposure to the inhibitor. After ~10 min, 2-10 µM chelerythrine and 100 nM staurosporine decreased whole cell currents to 24 ± 18% (range 0-41%; n = 3) and 30 ± 20% (range 0-53%; n = 4), respectively. In two control experiments in which DMSO (0.05% vol/vol) was used, channel activity spontaneously decreased to 71% (range 60-81%) of the initial activity.


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Fig. 4.   Effect of chelerythrine on whole cell currents carried by 5 mM Ca2+ and recorded using perforated patch-clamp configuration. A: current-voltage relationship from 2 experiments (squares vs. circles) before (open symbols) and 10 min after (filled symbols) exposure to 2 µM chelerythrine. Inset: example of leak-subtracted currents elicited by step pulses to +10 mV. B: time course of chelerythrine and staurosporine effect on whole cell currents measured at +20 mV; bullet , 2 experiments with exposure to vehicle at time 0; open circle , 3 experiments with exposure to 2 µM (n = 2) and 10 µM (n = 1) chelerythrine at time 0; star , 2 experiments with exposure to 100 nM staurosporine at time 0.

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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Fig. 5.   A: effect of 50 nM (n = 3) and 100 nM (n = 8) staurosporine on Ca2+ channel activity. Because changes in channel gating using both concentrations were similar (not shown), data from all experiments were averaged. B: effect of high-K+, low-Ca2+ solution alone (n = 4) and in presence of DMSO (0.05% vol/vol; vehicle; n = 6) on Ca2+ channel activity. All control experiments were averaged, since results from both solutions were similar. Solid lines, presence of staurosporine (A) and DMSO (B). Dashed lines, control window and second window in presence of staurosporine (A) and DMSO (B). Means ± SE are shown.

Figure 6A shows the time course of one experiment in which two channels were present in the patch. Figure 6B compares 33 control sweeps obtained before and ~6 min after exposure to staurosporine. It is noteworthy that the main effect of staurosporine was to decrease the number of sweeps showing channel openings (active sweeps). In 11 experiments, the fraction of active sweeps decreased from 0.47 ± 0.30 in the control window to 0.15 ± 0.17 during the second window after addition of staurosporine (P < 0.01, paired t-test). On the other hand, the fraction of active sweeps was not affected by the vehicle or prolonged exposure to the high-K+, low-Ca2+ solution. The fraction of active sweeps in the control and second window were 0.40 ± 0.28 and 0.40 ± 0.29, respectively ( P = not significant, paired t-test).


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Fig. 6.   A: diary of NPo showing effect of 100 nM staurosporine in a patch with 2 channels. Dots indicate presence of null sweeps. Dotted lines at top indicate sweeps shown in B. Arrow, time drug was added. B: 33 sweeps recorded before (control; left) and after (right) 6 min of exposure to staurosporine. Currents were elicited by step pulses from a holding potential of -40 mV to +10 mV (patch b3722).

These experiments suggests that staurosporine decreased channel availability because step pulses were sufficiently long to permit opening of all available channels, and we observed no change in first latency distributions (see Fig. 7C). Under an assumption of independent channel gating (1), the probability that a channel opens upon maximal depolarization (Pavail) was calculated from the fraction of null sweeps using binomial statistics (Eq. 1), where N is the number of channels in the patch (22). In our measurements, Pavail may be overestimated, since N is a low estimate of the channels in the patch (16)
Null sweeps/total sweeps = (1 − <IT>P</IT><SUB>avail</SUB>)<SUP><IT>N</IT></SUP> (1)
Staurosporine significantly decreased Pavail from 39 ± 21% in the control window to 11 ± 12% during the second window after addition (P < 0.01, n = 11). On the other hand, the depolarizing solution, in the presence or absence of the vehicle, did not affect channel availability, since Pavail was similar in the control window (35 ± 20%) and during the second window after addition (24 ± 16%) (not significant, n = 10).


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Fig. 7.   Effect of staurosporine on mean open time distribution (A and B), cumulative first latency distribution (C), and single-channel amplitude-duration relationship (D) from 11 experiments. This analysis was carried out with events recorded during control window and second window in presence of staurosporine. A and B: dashed lines fitting histograms for control (A) and staurosporine (B) indicate single-exponential components, whereas solid lines indicate sums of these functions. Fitted parameters are indicated in text. C: cumulative first latency distribution from 4 experiments in which more than 1 channel was present in patch. D: single-channel amplitude-duration relationship from 11 experiments. Single open events were recorded during control window (1,122 events; open circles) and after exposure to 50-100 nM staurosporine (170 events; shaded circles). Only open events >0.53 ms were used to calculate single-channel currents, which amount to 0.79 ± 0.14 pA in control and 0.74 ± 0.13 pA in staurosporine.

Figure 7 shows the channel open time distribution calculated from events recorded during the control window (A) and during the second window in the presence of staurosporine (B). The open time distribution in control was fitted to three exponential functions with time constants tau 1 = 0.3 ms, tau 2 = 0.7 ms, and tau 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 tau 1 = 0.2 ms and tau 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 chi 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).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 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 alpha 1-subunit in aortic cells (38) but increases the content of PKCalpha (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 beta -adrenergic agonists (13, 14) and the observed shift between gating modes (42). At variance with the effect of beta -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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