Abstract Modulation of the inward Na+ current (INa) by protein kinase C (PKC) was investigated by intracellular perfusion of a peptide corresponding to the catalytic subunit of PKC (PKCP). The effects of PKC activation independent of membrane-receptor pathways were studied in neonatal rat ventricular myocytes using whole-cell patch-clamp techniques. Perfusion with 2 nmol/L PKCP caused a depolarizing shift in steady state half-inactivation relative to control (−83.2±1.3 versus −74.9±1.6 mV for control versus PKCP, respectively) without a change in current-voltage relationships or peak INa. The development of resting inactivation was slowed by PKCP (τ, 69.1±7.6 [control] versus 100.4±5.1 ms). Open-channel inactivation, estimated by measuring INa decay from peak current at test voltages between −10 and +30 mV was significantly slowed by PKCP. Recovery from inactivation was more rapid during PKCP perfusion, with a shortening of both the fast (τf) and slow (τs) components of τ (τf, 38.5±7.0 [control] versus 14.2±4.7 ms; τs, 163.4±47.9 [control] versus 51.3±9.2 ms). All of the effects of PKCP on INa were antagonized by the PKC inhibitors chelerythrine chloride or staurosporine or by downregulation of PKC using phorbol ester preincubation. We conclude that the actions of PKC on the Na+ channel result in slowing the development of inactivation and accelerating reactivation, resulting in less resting inactivation.
The rapid inward INa initiates the action potential, making it a major determinant of cardiac conduction. Like many ionic currents, INa amplitude and kinetics can be modulated by second messenger systems, including phosphorylation pathways. This may be of considerable importance during ischemic events, when several intracellular signaling systems are activated, such as PKC.
Previous studies of the effects of PKC on INa amplitude and kinetics in neonatal rat cardiac myocytes have been contradictory. Maximal INa reportedly decreases after PKC stimulation with phorbol ester or diacylglycerol analogues,1 2 although others using the same agonists have recorded an increase of INa.3 The effects on inactivation kinetics also vary, with studies showing slowing,2 acceleration,3 or no change of the time course of inactivation.1 The diversity of responses observed suggests that multiple pathways are activated with the agonists studied. This is not surprising, because phorbol esters are not specific for PKC and have receptor sites on cytoplasmic proteins with other cellular actions, such as η-chimerin4 and phospholipase D.5 6 7
To avoid non-PKC effects that may be associated with activation of nonspecific membrane receptor pathways, we examined the effects of PKC activation on cardiac INa in intact cells using PKCP.8 This peptide activates PKC by blocking the autoregulatory pseudosubstrate region of native PKC, thereby exposing the active site and allowing substrate phosphorylation. Since the peptide acts directly on endogenous PKC, its effects on INa can be attributed specifically to PKC activation.
Direct PKC activation may have several sites of modulation of INa. There have been reports of multiple phosphorylation sites on the Na+ channel.9 One of these sites, serine1506, located in an intracellular loop linking domains III and IV of the α subunit, functions in inactivation10 and is reputed to be phosphorylated solely by PKC.11 However, PKC has a diverse array of phosphorylation substrates within the cell, including cytoskeletal attachment proteins.11 Since cytoskeletal continuity with the sarcolemma may also influence INa,12 phosphorylation of a protein other than the channel itself may also modulate INa. In the present study, we describe the effects of activated PKC on INa in intact cells.
Materials and Methods
Isolated ventricular cells were prepared from the hearts of 1-day-old Sprague-Dawley rats by enzymatic dissociation.13 The rat pups were decapitated, and the hearts were excised and minced in a 0.9% saline solution. The tissue was dissociated in 50 mL of Joklik MEM supplemented with (mmol/L) HEPES 10, sodium pyruvate 10, nicotinamide 1, l-ascorbate 0.4, adenosine 1, d-ribose 1, MgCl2 1, taurine 1, dl-carnitine 2, KHCO3 26, and l-glutamine 5, pH 7.6, with 10 μg/mL gentamicin, 50 μmol/L CaCl2, and 0.5 mg/mL collagenase (Worthington Scientific). After six changes of the medium, the cells were centrifuged and suspended in medium enriched with fetal bovine serum, plated, and incubated at 37°C. For these studies, cells were used 18 to 36 hours after plating, after they had attached to the coverslip glass and before they lost their spherical shape. All recordings were performed at room temperature (20°C to 22°C).
The extracellular solution contained (mmol/L) NaCl 130, NaHCO3 5, KCl 5.4, CaCl2 1, MgCl2 1, CoCl2 1, glucose 10, and HEPES 5, adjusted to pH 7.2 with NaOH. CoCl2 was used to block T- and L-type ICa. Since it was present in both control and PKCP superfusions, any shift in activation caused by this divalent ion14 or voltage-dependent block of INa15 should be comparable between treatment groups. However, to ensure that cobalt had no effect on the modulation of INa by PKC, several experiments were performed substituting either 5 μmol/L bepridil or 1 mmol/L amiloride for 1 mmol/L CoCl2. In this concentration, amiloride has been shown to block T-type ICa16 without significantly affecting INa.17 Bepridil, a nonspecific Ca2+ channel blocker,16 was dissolved in DMSO and added to the bath solution such that the concentration of DMSO in the bath was <1:10 000. V1/2 of activation, assessed in these cobalt-free solutions, was not significantly different from that in the cobalt-containing solutions either in the control condition or with PKCP in the pipette (V1/2, 39.7±1.8 mV [control, n=5] and 42.1±2.3 mV [PKCP, n=4]).
The pipette solution contained (mmol/L) CsCl 120, CsOH 5, NaCl 5, MgCl2 5, EGTA 1, Na2-ATP 5, and MOPS 10, adjusted to pH 7.2 with CsOH. In staurosporine-inhibition experiments, ATP was removed from the pipette solution to avoid competition at the ATP binding site on PKC.18
PKC catalytic subunit (Sigma Chemical Co) was dissolved in 0.05 mol/L acetic acid stock solution and frozen. PKCP (2 to 4 nmol/L) from this stock solution was added to the pipette solution the day of the experiment. The final dilution of the acetic acid stock was 1:10 000, and the measured pH of the pipette solution (pH 7.2) did not differ from control. PKC was inhibited by superfusion of antagonists dissolved in extracellular solution. Chelerythrine chloride, PMA, and staurosporine were dissolved in DMSO in concentrations that allowed the DMSO concentration to remain at <1:10 000 in all experiments. Control cells were exposed to the same concentration of DMSO. In addition, a comparison of cells studied in the presence or absence of DMSO revealed no effect on the kinetics and amplitude of INa, indicating that these concentrations of DMSO had no direct effect on the measured current.
Borosilicate glass capillaries (World Precision Instruments, Inc) were used to make pipettes using conventional pulling techniques. Electrode resistance in extracellular solution ranged from 1 to 4 MΩ. Whole-cell currents were recorded with an Axopatch 200A amplifier (Axon Instruments), as described previously.19 Series resistance (<10 MΩ) and cell capacitance were compensated electronically, and voltages were corrected for the liquid junctional potential. Average cell capacitance was 9.6±0.19 pF (n=58). Seal resistances were 1 to 2 GΩ. Data were filtered at 5 kHz, sampled at 20 Hz, and saved on floppy diskettes for off-line analysis.
The whole-cell voltage clamp was performed using the suction pipette method.20 After access to the cell interior had been gained, 2 minutes was allowed for equilibration of the pipette solution with the cytoplasm before electrophysiological recording began. Since PKCP was in the pipette solution, data for PKCP effects on INa came from cells different from control. Inhibition experiments were performed either by preincubation with the inhibitor or by wash-in of the inhibitor after control recordings. The type of inhibition is noted in the results.
Current-voltage relationships were determined in all cells before and after any other experimental protocols to test the adequacy of the voltage clamp. Activation was assessed with 20-ms pulses from a holding potential of −100 mV to test potentials between −80 and +70 mV. Data from cells were discarded if series resistance changed by >10% over the course of the recordings21 or if the slope of activation was <5.5 mV, because this indicates inadequate voltage control.22 The mean slope factor under these conditions was ≈7 mV (see “Results”). Of note, poor voltage control was noted in <10% of cells. To verify that adequate voltage control was achieved in normal [Na+]o, control experiments were conducted in reduced [Na+]o (68 mmol/L, n=6). The mean slope factor (7.8±0.6 mV, n=6 ) was similar to the values in 135 mmol/L Na+, as was the V1/2 of activation.
Steady state inactivation was determined using a standard two-pulse protocol, with a holding potential of −100 mV and a test potential of 0 mV. One-second conditioning pulses were applied 2 ms before the test potential to assess INa availability. Reactivation was also assessed using a two-pulse protocol. A 200-ms conditioning pulse was followed at varying intervals by a 20-ms test pulse from −90 or −110 mV to 0 mV. The development of resting inactivation was measured using a two-pulse protocol with a conditioning pulse of variable duration (1 to 200 ms) to −65 or −75 mV, followed by a 20-ms test pulse to 0 mV.23
Stimulation protocols, data collection, and analysis were performed using pClamp software (Axon Instruments). All data are expressed as mean±SEM, and comparisons between treatment groups were evaluated by Student’s t test or, where appropriate, an ANOVA followed by a Newman-Keuls post hoc test. A value of P<.05 was considered significant.
Current-voltage relationships were assessed in all cells. They were minimally affected by PKCP. INa was maximal at −27.5±1.8 mV in control and −27.3±2.0 mV with PKCP perfusion, and reversal occurred near the calculated Na+ equilibrium potential of 57 mV (Fig 1⇓). All active current was blocked by 50 μmol/L tetrodotoxin, confirming that INa was being measured in isolation. Conductance transformations of peak currents were fit with a Boltzmann equation to better describe the activation process. There were no significant changes of either half-activation (−42.1±1.2 versus −38.9±1.4 mV, P=NS) or slope factor (6.9±0.3 versus 6.3±0.4 mV, P=NS; control=22 cells, PKCP=19 cells) with PKCP perfusion.
To determine if peak current amplitude in the absence of resting inactivation was affected by PKC stimulation, we measured INa with depolarizations to 0 mV from a more hyperpolarized holding potential of −125 mV. INa was assessed periodically during the recording to evaluate temporal changes in current amplitude. No decrease of INa was noted in control cells, confirming the lack of current rundown in this preparation.19 There was also no significant decline in peak current amplitude normalized for cell capacitance during PKCP perfusion (254.6±17.8 versus 238.3±13.9 pA/pF, P=NS; control=15 cells, PKCP=14 cells).
Although there were no effects of PKCP on INa activation, voltage-dependent current availability was significantly altered. During PKCP perfusion, steady state half-inactivation shifted 8 mV in the depolarizing direction (−83.2±1.3 versus −74.9±1.6 mV, P<.001; control=12 cells, PKCP=15 cells). A comparison of mean steady state inactivation is shown in Fig 2A⇓. This effect increased in a dose-dependent manner: intracellular perfusion with 4 nmol/L PKCP caused a further depolarizing shift in steady state inactivation (−72.4±1.8 mV, n=5). However, 100 nmol/L PKCP caused only an additional 3-mV depolarizing shift (−69.4 mV, n=2), indicating that 4 nmol/L is close to a saturating concentration. In two additional cells, the effect of 2 nmol/L PKCP on steady state inactivation was assessed in cobalt-free solutions. There was a 10-mV depolarizing shift of half-inactivation, which was similar to that measured when cobalt was used to block ICa.
The effect of PKCP on current availability reached steady state within the several minutes necessary to characterize activation and the adequacy of voltage clamp. As reported previously, no systematic temporal shifts of steady state inactivation were observed in neonatal cells.19 To reconfirm this finding, sequential measurements of steady state inactivation were conducted in 47 cells. Small hyperpolarizing (27 cells) or depolarizing (20 cells) shifts occurred over the average 10-minute recording time. However, the mean V1/2 of inactivation in these cells changed <1 mV over the duration of the recording. We presume that the stability of voltage-dependent current availability in these small cells results from intracellular equilibrium achieved during the 2-minute period before recording.
The voltage dependence of inactivation was shallower during PKC stimulation, as evidenced by an increased slope factor of curves fit with a Boltzmann equation (−5.4±0.2 versus −6.2±0.3 mV, P=.007; control=12 cells, PKCP=15 cells). The depolarizing voltage shift and change of slope factor both contribute to the presence of more recruitable current at depolarized membrane potentials (Fig 2B⇑ and 2C⇑). The change in slope of the steady state inactivation curve could be due to an increase in slow inactivation during the 1000-ms conditioning pulse rather than an actual change in voltage dependence. To assess this possibility, the slopes of steady state inactivation curves were measured using 5000- and 1000-ms conditioning pulses in the same cell. The longer conditioning pulse duration should result in more slow inactivation. However, there was no change in slope factor associated with the increase in conditioning pulse duration either in the control cells (−5.8±0.5 versus −5.7±0.6 mV, P=NS; n=3) or in the PKCP-treated cells (−6.3±0.6 versus −6.6±0.6 mV, P=NS; n=5). This suggests that PKCP causes a true change in the voltage dependence of steady state inactivation.
To ensure that PKCP was indeed activating PKC in these cells, a specific PKC inhibitor, chelerythrine chloride,24 was included in the bath solution. The effect of PKCP on steady state inactivation was reversed when the extracellular solution contained 10 μmol/L of chelerythrine chloride (−81.1±1.4 mV, n=4). The depolarizing shift of resting inactivation induced by PKCP was also prevented by two other PKC inhibitors with molecular mechanisms different from chelerythrine chloride. Downregulation of PKC by preincubating the cells in 100 nmol/L PMA overnight25 (V1/2=−86.1±1.1 mV, n=3) and superfusion with 100 nmol/L staurosporine (V1/2=−83.6±2.3 mV, n=5) both inhibited the effect. Because the molecular action of staurosporine inhibition is competitive binding at the ATP binding site on PKC, ATP was eliminated from the pipette solution for the experiments with staurosporine. This did not cause a change in steady state inactivation in control cells (−82.2±0.04 mV, n=3). Staurosporine or chelerythrine chloride applied to control cells in the absence of PKCP also failed to change current availability (−83.4±1.3 mV, n=3). Therefore, the effect of these inhibitors was specifically the inhibition of PKCP.
The time course of the development of inactivation was studied with a double-pulse protocol, with conditioning pulses of varying duration to −65 mV followed by a test potential of 0 mV. No macroscopic current is observed with depolarizations to −65 mV in control conditions or with PKC, so this allows assessment of direct transitions from the resting to the inactivated state.23 26 The onset of inactivation is fit well with a single-exponential equation (Fig 3⇓). PKCP prolonged the time constant of this process at −65 mV (69.1±7.6 versus 100.4±5.1 ms, P=.004; control=6 cells, PKCP=8 cells). The development of inactivation was also assessed from a more hyperpolarizing holding potential of −75 mV in several experiments. At this potential, there was a trend toward an increase of time constant of the development of inactivation with PKCP (76.7±9.9 versus 101.6±9.0 ms, P=.06; control=6 cells, PKCP=3 cells). This modulation of INa gating characteristics is absent when PKC is inhibited by chelerythrine chloride (80.3±2.8 ms, n=2) or PMA incubation (75.5 ms, n=1).
To assess further the possibility that PKCP was contributing to slow development of inactivation, the onset of resting inactivation at −65 mV was measured using the same two-pulse protocol with a conditioning pulse varying in duration from 1 to 1000 ms. With this conditioning pulse interval, the process is best fit with a double-exponential equation. PKCP prolongs each of the time constants (τf=42.2±6.4 versus 63.1±10.7 ms, P=.04; control=7 cells, PKCP=7 cells) and (τs=145.9±16.2 versus 291.1±81.8 ms, P=.03) without affecting the proportion that each contributes to recovery (Af=0.62±0.07 versus 0.65±0.09, P=NS).
Given that the time course of resting inactivation was slowed by PKC, the time course of the decay of INa was measured as a macroscopic estimation of the development of inactivation from the open state. For these experiments, pulses from −100 mV to test potentials between −30 and +30 mV were applied to the cells, and the time constants of decay were measured. INa decay is a complex process in these cells, best fit as a double exponential at negative potentials but a single exponential at positive potentials. The goodness of fit was determined visually and by the standard deviation of the fit. PKCP increased the decay time constants of INa at voltages positive to −10 mV but not at more negative potentials (Table⇓). Therefore, INa decay, within a physiologically relevant range, is also slowed by PKC activation.
As another measure of the open to inactivated state transition, we used a two-pulse protocol with a conditioning pulse (−30 mV) of 1- to 20-ms duration. Since this protocol measures available current after a conditioning pulse, it is less subject to contamination by delayed activation.27 Normalized current amplitudes were well fit with a double-exponential equation, as was INa decay at this potential. The time constants from this protocol were quantitatively similar to the INa decay time constants (τf=0.70±0.07 versus 0.86±0.12 ms, P=NS; control=7 cells, PKCP=7 cells; τs=5.02±0.75 versus 5.51±0.83 ms, P=NS). τf accounted for 70% of the current amplitude, suggesting that at −30 mV the predominant transition measured is open to inactivated states. At this test potential, PKCP did not delay the open to inactivated transition or delay activation.
At a more negative voltage (−45 mV), two time constants are again noted (τf=2.13±0.34 versus 2.22±0.40 ms, P=NS; control=5 cells, PKCP=7 cells; τs=22.6±6.0 versus 41.4 ms, P=.03), with τf predominating (65±2% versus 75±3%, P=.01). Of note, τs is prolonged significantly by PKCP.
The finding that the transitions into inactivated states are slowed with PKC stimulation suggests that phosphorylation of the channel itself or a channel-associated protein may slow channel gating in general. If so, then the time course of transitions out of the inactivated state would also be prolonged. Alternatively, PKC stimulation may cause the inactivated states to be less thermodynamically favored; in which case, transitions out of this state would be accelerated. To distinguish these possibilities, we measured reactivation, ie, the transition from inactivated to closed states at normal (−90-mV) and hyperpolarized (−110-mV) membrane potentials. At both potentials, reactivation is best described as a double-exponential process. Recovery from inactivation was significantly faster during PKCP perfusion, and both time constants were decreased (Fig 4⇓). At −90 mV, τf decreased from 38.5±7.0 to 14.2±4.7 ms (n=11, P<.002), whereas τs decreased from 163.4±47.9 to 51.3±9.2 ms (P=.002). The acceleration of INa recovery caused by PKC was reversed by chelerythrine chloride (τf=30.7±12.7 ms, τs=199.3±72.1 ms; n=2), by PMA treatment (τf=44.3 ms, τs=107.3 ms; n=1), and by staurosporine (τf=31.7±8.1 ms, τs=149.9±38.7 ms; n=3). At membrane voltage of −110 mV, PKCP decreased τf from 10.5±1.4 to 6.1±1.0 ms (n=16, P=.02) without a significant change in τs.
This is the first report of INa modulation by direct stimulation of the PKC protein by perfusing a catalytic subunit of PKC into the intracellular space. Specificity of PKCP was verified by three chemically distinct PKC inhibitors. Chelerythrine chloride acts at a site on the native catalytic subunit but does not compete with ATP,24 whereas staurosporine inhibits PKC by occupying the ATP binding site.18 Finally, preincubating with the phorbol ester PMA downregulates native phorbol ester–sensitive PKC, which also prevented the actions of PKCP. All three PKC inhibitors effectively reversed or prevented the shift in steady state inactivation, the slowed development of inactivation, and the accelerated recovery from inactivation. We conclude from this that PKCP was activating endogenous PKC and that the changes in INa kinetics recorded were a consequence of PKC activation.
The catalytic domain of PKC has a high degree of homology among isoforms,28 theoretically allowing activation of multiple PKCs. However, the inhibition of PKCP by phorbol ester downregulation suggests that changes in INa reported in the present study are caused by the phorbol ester–responsive classes of PKC, the conventional and novel isoforms of PKC.
The primary action of PKC stimulation was to modulate the time course of state transitions. The development of inactivation, from rest and open states, was slowed with PKCP perfusion. Interestingly, the time constant of inactivation from resting states in control cells was longer at −75 than at −65 mV but not in PKCP-perfused cells, implying that PKCP may have changed the voltage dependence of inactivation from resting states. Conditioning pulses to −45 mV, a voltage at which inactivation can develop from both open and closed states, results in a family of currents that decay with two time constants. τf is quantitatively similar to time constants of INa decay at that voltage, whereas τs is similar in magnitude to that measured from resting states. Only τs is prolonged by PKCP. If it is presumed that τs represents a single-channel transition, this would mean that PKCP alters inactivation from resting states across a voltage range (−75 to −45 mV). However, whole-cell voltage-clamp protocols cannot exclude the possibility of multiple or delayed openings, which would imply alternate interpretations of these data, including modulation of the back transition rate by PKCP.
In contrast, the recovery from inactivation (ie, reactivation) was accelerated. The dual-exponential time course of reactivation implies multiple inactivated states. The relative proportion of recovery by the two time constants was unaffected by PKCP, suggesting that PKC stimulation does not promote the filling of faster reactivating inactivated states. Rather, the residence time in both inactivated states is reduced by phosphorylation. The net result of slowing the development of inactivation and accelerating reactivation is to decrease the probability of channels being inactivated, causing an increase in INa availability.
PKC activation by PKCP shows similarities to PKC activation by phorbol esters. Decay of INa was slowed at voltages between −10 and 30 mV, which agrees with a change in kinetics described after phorbol ester stimulation with OAG2 29 but not with TPA.3 Although both OAG and TPA should activate the same isoforms of PKC by acting at the phorbol ester binding site on the regulatory subunit, the modulatory effects of these substances on INa are clearly distinct.2 3 29 30 These opposing effects may be due to activation of different PKC isoforms31 30 or to non–PKC-mediated effects of phorbol esters, such as phospholipase D activation. The slowed development of inactivation from closed states, observed in the present study, is in contrast to the effect of diacylglycerol seen in neuroblastoma cells.32 No effect on INa amplitude was observed with PKC stimulation for up to 15 minutes, in contrast to several previous studies. A decrease of INa in response to OAG has been reported in neonatal rat heart,1 2 and an increase in INa amplitude has been reported in response to the phorbol ester TPA.3 If these opposing effects are due to activation of different PKC isoforms, then the lack of response noted in our experiments is likely due to activation of isoforms with opposing actions. Alternatively, shifting of the activation and inactivation curves could cause changes of INa amplitude, depending on the test and holding potentials chosen.
It has been proposed that PKC phosphorylates a site on an intracellular loop of the α subunit that affects inactivation.33 Consistent with this hypothesis, the major effects reported by the present study involved changes in either the amount of resting inactivation or in the kinetics of transit into and out of inactivated states. Although these findings are consistent with direct phosphorylation of the Na+ channel, it is also possible that phosphorylation of an endogenous channel-associated protein or cytoskeletal element could also cause the modulation of INa observed. Taken together, our data suggest that in intact cells, stimulation of PKC causes the inactivated state conformations to be less thermodynamically favorable, so that the amount of time spent in the inactivated states is reduced. However, direct recordings of single-channel transitions are necessary to test this hypothesis.
The PKC isoforms expressed in neonatal rat cardiomyocytes are the same as in the adult rat, with the addition of the ζ isoform.31 Furthermore, the effects of phorbol esters on INa in neonatal cardiac cells1 2 3 are very similar to those observed in the stably expressed rH1 α subunit of the channel.2 The PKC catalytic subunit fragment used to stimulate native PKC is structurally specific to PKC, being a pseudosubstrate for the autoregulatory subunit. Internal perfusion of PKCP, activating PKC downstream from the receptor and associated G proteins, allowed us to quantify PKC effects on INa in isolation from known G-protein modulation of INa.34
Our results must be interpreted in light of certain limitations. Perfusion of the cell interior may have affected some soluble cytoplasmic elements that contribute to the regulation of INa. However, the complete reversal of the modulatory effects of PKC on INa by PKC inhibitors implies that intracellular dialysis did not have a profound effect. From these studies in whole cells, it is not possible to discriminate between INa modulation due to PKC phosphorylation of the Na+ channel or to phosphorylation of other regulatory proteins that subsequently affect INa. However, the rapid response noted with PKCP perfusion suggests that complex intermediate steps are not involved. Moreover, since multiple phosphorylation events are possible in vivo, the PKCP effects measured with whole-cell recordings more closely reflect INa modulation under physiological conditions. Finally, rate constants of channel state transitions were not measured; they were only estimated from the recording of macroscopic currents. For instance, delayed openings or multiple openings may have affected the observed inactivation time constants. However, the holding and test voltages were chosen so that the time constants would measure the transit between two predominant channel states.
The modulation of INa during PKC stimulation may have important physiological effects on cardiac function. INa is a major contributor to the upstroke of the cardiac action potential and thus ventricular conduction. Since V1/2 of steady state inactivation lies close to the resting membrane potential, small shifts of inactivation or resting potential can have profound effects on the amplitude of the action potential upstroke.35 The depolarizing shifts of inactivation noted with PKC stimulation will tend to offset the decrease in INa caused by other cellular changes associated with myocardial ischemia and infarction, such as membrane depolarization and acidosis. Thus, PKC activation may serve as a homeostatic mechanism to preserve cardiac conduction during ischemic stress.
Selected Abbreviations and Acronyms
|τf, τs||=||fast and slow time constants|
|Af||=||fraction of current recovery with τf|
|bepridil||=||B-[(2-methylpropoxy)methyl]-N-phenyl-N-(phenyl-methyl)-1-pyrrolidineethanamine, HCl salt|
|I Ca||=||Ca2+ current|
|I Na||=||Na+ current|
|PKC||=||protein kinase C|
|PKCP||=||peptide analogue of the PKC catalytic region|
|PMA||=||phorbol 12-myristate 13-acetate|
|V 1/2||=||half-(in)activation voltage|
This study was supported by a grant from the American Heart Association, Maryland Affiliate.
- Received January 21, 1997.
- Accepted June 9, 1997.
- © 1997 American Heart Association, Inc.
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