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(Circulation Research. 1997;80:370-376.)
© 1997 American Heart Association, Inc.

Articles

Functional Effects of Protein Kinase C Activation on the Human Cardiac Na⁺ Channel

Katherine T. Murray, NingNing Hu, J. Richard Daw, Hyeon-Gyu Shin, Marshall T. Watson, Amy B. Mashburn, Alfred L. George, Jr

the Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The cardiac Na⁺ current plays an important role in determining normal and abnormal impulse propagation in the heart. We have investigated the effects of protein kinase C (PKC) activation on the recombinant human cardiac Na⁺ channel (hH1) following heterologous expression in Xenopus laevis oocytes. Phorbol 12-myristate 13-acetate (PMA), which directly activates PKC, reduced current amplitude at all test potentials (43±12% at -10 mV). In contrast to the rat brain IIA (rBIIA) channel, there was no apparent change in either macroscopic Na⁺ current decay or the voltage dependence of channel gating. Further experiments indicate that the effects of PMA were mediated by PKC activation: (1) an inactive stereoisomer, 4-PMA, had no effect; (2) preincubation with the protein kinase inhibitor chelerythrine prevented the PMA effects; and (3) a hydrolyzable diacylglycerol analogue, 1-oleoyl-2-acetyl-glycerol, also reduced current (22±5%). In addition, when the _1B-adrenergic receptor was coexpressed with hH1, the -receptor agonist methoxamine reduced hH1 current (45±10%), an effect that could be eliminated by chelerythrine preincubation. When a conserved consensus PKC site (serine 1503) in the III-IV interdomain linker thought to be responsible for the PKC effects on rBIIA was mutated, PMA still reduced Na⁺ current, but the magnitude of the effect was smaller compared with that for the wild-type channel. Similar findings were obtained with ₁-receptor stimulation following receptor coexpression with the mutant channel. We conclude that activation of PKC modulates the human cardiac Na⁺ channel by at least two mechanisms, one similar to that seen with rat brain channels, involving a conserved putative PKC site, and a second more specific to the cardiac isoform.

Key Words: sodium channels • heart • phosphorylation • protein kinase C • xenopus oocyte

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The function of voltage-gated Na⁺ channels is a critical determinant of conduction in the heart. There is substantial evidence that abnormal function of the Na⁺ channel can play an important role in the genesis of life-threatening cardiac arrhythmias by at least two mechanisms. First, mapping studies have shown that sustained ventricular tachyarrhythmias often occur because of a reentrant mechanism.^{1 2} In this circumstance, slowed conduction due to reduced Na⁺ current facilitates the development of arrhythmias based on a reentrant circuit. Second, mutations in the cardiac Na⁺ channel can result in abnormal repolarization and associated serious arrhythmias in some patients with the congenital long-QT syndrome.^{3 4} Thus, the factors that alter Na⁺ channel function on a transient or permanent basis could have important clinical implications.

The adrenergic nervous system has long been recognized as an important modulator of cardiac electrophysiology.⁵ Adrenergic stimulation, which activates cAMP-dependent protein kinase (PKA) and PKC, can facilitate cardiac arrhythmias in animal models and humans (although the effects of -receptor stimulation are somewhat controversial).^{5 6 7} Previous studies to investigate the effects of kinase activation on Na⁺ current in native cardiac myocytes have provided conflicting results.^{8 9 10 11 12 13} Activation of either PKA or PKC has been reported to cause an increase in Na⁺ current in some studies, a reduction of current in others, and variable shifts in the voltage dependence of channel gating.

A human cardiac-specific voltage-gated Na⁺ channel (hH1) isoform has been cloned.¹⁴ Expression of hH1 results in a current with kinetics, voltage dependence of channel gating, and pharmacology consistent with it being the major tetrodotoxin-resistant Na⁺ channel isoform in human heart. Multiple consensus sites for phosphorylation are present in the hH1 sequence, particularly for PKC. However, the factors that regulate function of this channel have not been fully characterized.

In previous investigations, the effects of PKC stimulation on recombinant rBIIA have been studied extensively.^{15 16} These effects include a reduction in current amplitude and slowing of macroscopic current decay, which reflects the rate of channel inactivation. On the basis of mutagenesis experiments, these effects were attributed to phosphorylation of a PKC consensus site in the III-IV interdomain linker, serine 1506.¹⁶ Although this conserved consensus site is also present in the hH1 sequence (serine 1503), less than a third of the multiple other PKC sites present are conserved between the brain and cardiac isoforms.

In these experiments, we have used the human cardiac Na⁺ channel cDNA to test the hypothesis that the human cardiac Na⁺ current is modulated by PKC activation. In addition, we have mutated the conserved PKC site in the III-IV interdomain linker to probe the molecular mechanism of effect. Our results indicate that stimulation of PKC has important functional consequences for hH1, with a molecular basis that is distinct from that described for other Na⁺ channels.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental Preparation
Xenopus laevis frogs were purchased from Xenopus 1 (Ann Arbor, Mich). Oocytes were removed and disaggregated by exposure to collagenase (1 mg/mL, type II, Worthington) as previously described.¹⁷ The cells were placed in a physiological solution (ND96; see below) and incubated at room temperature for 1 to 24 hours before cytoplasmic RNA injection. Sense cRNA was transcribed in vitro in the presence of the methylated 5'-cap analogue m⁷GpppG from the Xba I–linearized pSP64T-hH1 template using SP6 RNA polymerase. Using RNase-free water, the RNA was diluted before injection, so that Na⁺ currents expressed within 24 to 48 hours were <5 µA when 30 to 35 nL was injected. The oocytes were kept at 20°C for 1 to 4 days until experimentation. Cells from each isolation were screened for endogenous Cl^- currents using depolarizing voltage-clamp steps (-110 to +40 mV). Oocytes having endogenous currents at these voltages, which were >1% of expressed currents, were not used. Hamster _1B-adrenergic receptor cDNA¹⁸ in the SP65 vector was kindly provided by Dr Robert Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC). The template was linearized with Xba I, and cRNA was generated using the SP6 polymerase. Expression of the _1B-receptor was verified by the development of an inward current after administration of an -adrenergic receptor agonist, methoxamine, 24 to 48 hours after injection of cRNA diluted 1:1 with RNase-free water.¹⁹ During coexpression experiments with hH1, undiluted _1B-receptor cRNA was mixed in a 1:1 ratio with hH1 cRNA to generate a concentration based on initial studies known to generate appropriate-sized currents.

Electrophysiological Measurements
Oocytes were voltage-clamped using the two-microelectrode voltage-clamp technique as previously described.^{17 20 21} Microelectrodes were pulled from borosilicate glass (Radnoti Glass Technology, Inc, or Warner Instruments) and filled with 3 mol/L KCl. Tip resistances were 1.0 to 1.5 M for the current-passing electrode and 2 to 5 M for the voltage-measuring electrode. Membrane potentials were controlled by a high-compliance voltage-clamp amplifier (Clampator, Dagan), with data sampled at 80 kHz and filtered at 5 kHz. Voltage command potentials were generated by a 12-bit digital-to-analog converter controlled by customized pCLAMP software (Axon Instruments). A grounded metal shield was placed between the two electrodes to reduce capacitive coupling and to improve the speed of the clamp. Because voltage control of currents with rapid kinetics such as hH1 can be difficult in oocytes, we studied only those cells in which specific criteria for voltage control of Na⁺ currents were satisfied, as previously described.²² These included a smooth current contour, a 30- to 40-mV range from threshold to maximum peak current during determination of the current-voltage relationship, and a similar time-to-peak current during the test pulses of an inactivation curve.

The holding potential was -120 mV to allow full recovery from inactivation. Under control conditions, recovery from inactivation was complete in 500 milliseconds. Therefore, voltage-clamp pulses were delivered at 1-second intervals unless otherwise noted. Current recordings were obtained after electrode impalement at 30-second intervals using a fixed test pulse (-10 mV) for 10 minutes to ensure cell stability; Na⁺ current was also monitored in this way after the addition of a test compound to observe changes in current amplitude. The voltage-clamp protocols conducted under control conditions were repeated after changes in current amplitude approached steady state (see "Results"). To calculate cell membrane electrical capacitance, the capacitative transient was recorded during a small voltage step (-120 to -110 mV) during which hH1 current was not activated. Integration of the leak-corrected transient yielded the charge (Q) transferred during the voltage step (V) from which capacitance (C) was calculated: C=Q/V. All experiments were conducted at room temperature (22±2°C).

Solutions
A physiological solution (ND-96) was used for continuous superfusion of the oocytes; this solution contained (mmol/L) NaCl 96, KCl 2, CaCl₂ 1.8, MgCl₂ 1.0, and HEPES 5 (pH 7.5). The bath perfusion rate was 1 mL/min in all experiments. At this rate, there was up to a 5-minute delay for the experimental solution to reach the bath at the time of solution changes due to dead space. PMA, 4-PMA, and chelerythrine were obtained from LC Services, and methoxamine was purchased from Sigma.

Mutagenesis
The serine at position 1503 in the hH1 amino acid sequence was converted to an alanine (S1503A) using overlap extension polymerase chain reaction mutagenesis.^{23 24} The product was digested with Kpn I and BstE II and then directionally ligated into the Kpn I–BstE II site of the plasmid pSP64T-hH1. Multiple independent recombinant clones were analyzed by restriction digestion to verify correct assembly and to screen for the presence of the mutation (creation of a new Nar I site). The mutant inserts were fully sequenced, and clones lacking polymerase errors were chosen for electrophysiological studies.

Data Analysis
Analysis of the data was performed using custom programs that were designed to read and analyze pCLAMP data files. Leak subtraction was performed either on-line using the Clampator amplifier or by obtaining the least-squares fit to the passive linear leak current obtained during voltage steps from -120 to -80 mV, during which Na⁺ current was not activated. The time course of macroscopic current inactivation was fitted with an exponential function using a nonlinear least-squares algorithm. Inactivation curves were fitted with a Boltzmann equation. Measurements of peak current amplitude after administration of a test compound were normalized to predrug values using an average of five test pulses (over 2.5 minutes) immediately before drug superfusion. Student's paired t test was used to compare voltage-dependent and kinetic properties of hH1 current before and after the administration of a test compound. The effect of drug administration over time was analyzed by one-way ANOVA, with appropriate post hoc pairwise tests. A value of P.05 was used to reject the null hypothesis. Data are expressed as mean±1 SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In order to investigate the effects of PKC activation on the function of hH1, initial experiments were performed using the phorbol ester PMA at two different concentrations (50 and 10 nmol/L). Data are presented for the 10 nmol/L concentration, since similar results were obtained using the higher concentration, except where noted. Fig 1 demonstrates families of Na⁺currents in Xenopus oocytes expressing hH1 recorded during depolarizing voltage steps under baseline or control conditions (panel A) and after bath superfusion of PMA (panel B). It is evident that the primary effect of PMA was suppression of peak hH1 current at all test potentials examined. In this experiment, the reduction in peak Na⁺ current (at -10 mV) was 47% at 30 minutes of exposure and averaged 43±12% for the group of experiments using 10 nmol/L PMA (n=10). With 50 nmol/L PMA (n=4), the reduction in hH1 current was 71±13% after 30 minutes.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of PMA on hH1. A, Inward Na+ currents during step depolarizations in Xenopus oocytes injected with hH1 cRNA are shown under control conditions (pulse protocol in inset). B, Na+ currents from the same cell as in panel A are shown after exposure to 10 nmol/L PMA.

To further characterize the nature of this effect, we investigated whether PMA altered the voltage dependence of channel gating. The voltage range over which channel activation occurred was estimated by recording the voltage at which threshold (initial inward) and peak currents were obtained during the voltage-clamp protocol shown in Fig 1. There was a small shift of these values to more positive potentials, but the changes were not statistically significant (threshold/peak current was -50±2/-24±2 mV before and -41±3/-20±2 mV after 10 nmol/L PMA, respectively). We did not construct conductance-voltage plots for these experiments because of the uncertainty of measuring the reversal potential given the large capacitive transients and rapid Na⁺ currents seen in these cells. A standard two-pulse protocol was used to determine the voltage dependence of channel inactivation. Fig 2A shows averaged values for normalized steady state inactivation curves before and after the administration of 10 nmol/L PMA. There was no significant change in either V_½ or the k value of this curve after PMA (V_½ was -79±2 before and -80±3 mV after PMA, respectively; for k, similar values were 4.3±0.1 and 4.2±0.4). We also examined the time course of macroscopic Na⁺ current decay to determine the rate of channel inactivation, recognizing that these measurements during whole-cell recordings provide primarily an estimate of this process. Na⁺ current decay could be fit with a single-exponential function having a value of 0.7±0.1 milliseconds at -10 mV under baseline conditions. In contrast to rBIIA, there was no apparent change in the time course of inactivation of hH1 current with administration of 10 nmol/L PMA (=0.8±0.1 millisecond). A twin-pulse protocol was used to examine the effects of PMA on recovery of hH1 from inactivation. The time course of recovery is shown in Fig 2B and was best fit by a biexponential function. PMA did not alter either the fast or slow components of the recovery process (_fast /_slow=16±4/502±100 milliseconds before and =30±12/491±60 milliseconds after PMA 10 nmol/L).

View larger version (24K): [in this window] [in a new window]   Figure 2. Inactivation of hH1 current. A, A standard two-pulse protocol (inset) was used to assess the voltage dependence of steady state channel availability (inactivation curve) with initial 2-second prepulses varying from -140 to -30 mV, followed by a test pulse to a voltage producing maximal Na+ current (-10 or -20 mV). Data were normalized to peak current amplitude using a prepulse of -140 mV at baseline, and averaged values (mean±SEM) are presented. The line through the data represents the best nonlinear least-squares fit of a Boltzmann function. The half-inactivation voltage before () and after () the administration of PMA was -79±2 and -80±3 mV, respectively. Selected current tracings from a representative experiment are shown on the right. B, Recovery of hH1 current (at -120 mV) from inactivation before () and after () the administration of PMA was measured using a two-pulse protocol with an initial prepulse to permit development of inactivation (0 mV for 2 seconds), followed by a second test pulse (-10 or -20 mV) at different time points (recovery time) after the prepulse. Data were normalized to the maximum current value under control conditions at a recovery time of 20 seconds (data not shown) and were averaged. Na+ current tracings at different recovery times during an individual experiment are shown on the right.

The time course of PMA effect on hH1 Na⁺ current amplitude was examined in more detail by recording Na⁺ current every 30 seconds before and after the addition of PMA. Fig 3A shows the results of an individual experiment with peak current plotted as a function of time after electrode impalement of the oocyte. Peak Na⁺ current was normalized to the average value obtained before the addition of PMA to the bath superfusate (indicated by the arrow). Because previous studies have reported that phorbol esters can cause a concentration and time-dependent internalization of plasma membrane in oocytes,²⁵ we obtained simultaneous measurements of electrical capacitance (also shown normalized to predrug values) as a reflection of cell membrane surface area. In some experiments (Fig 3A), capacitance was unchanged despite a marked reduction in Na⁺ current amplitude; in others, there was a small reduction over time. To minimize any potential contribution of cell membrane internalization, we chose to analyze the data after 30 minutes of exposure to PMA (when effects on capacitance should be minimized²⁵ ) even though the reduction of current amplitude might not have reached steady state (potentially underestimating the full effect of PMA). Fig 3B shows summary data for normalized values of Na⁺ current and capacitance plotted at different time points after the addition of 10 nmol/L PMA. After 30 minutes, the reduction in hH1 current (43±12%) was significantly greater than the small decline in capacitance (11±7%). When current was normalized for capacitance, averaged hH1 current density fell from 13.9±2.5 nA/nF at baseline to 7.5±3.0 nA/nF after 30 minutes of PMA (a 46% reduction). These data indicate that although the reduction in hH1 current amplitude with PMA probably results from a combination of factors, it occurs to a major extent from modulation of channel function rather than internalization of channel-containing oocyte plasma membrane.

View larger version (26K): [in this window] [in a new window]   Figure 3. Time course of PMA effects. A, The time course of the effect of PMA on peak Na+ current at -10 mV () and capacitance () is illustrated for an individual experiment. Data are expressed normalized to the predrug value; either an average value from five test pulses over 2.5 minutes (current) or a single value (capacitance) immediately before drug superfusion was used. Time 0 corresponds to the time of oocyte impalement, and the arrow indicates the start of drug infusion into the bath. PMA reduced Na+ current in this experiment by 14% and 49% at 30 and 45 minutes, respectively, without a change in capacitance (+6 and +5%, respectively). B, Averaged values for peak Na+ current () and capacitance () are shown as a function of time after the addition of PMA at 10 nmol/L. Data were normalized to the value obtained just before the administration of PMA (t=0). There was a significant reduction in hH1 current of 43±12% at 30 minutes (*P.05). C, The inactive stereoisomer 4-PMA had no significant effect on hH1 current (-4±12% at 30 minutes, n=4). Current amplitude from each experiment was normalized to predrug values as in panel A. D, After pretreatment (30 minutes) with the PKC inhibitor chelerythrine (, 20 µmol/L), PMA administration had no effect on hH1 current (-5±7% after 30 minutes, n=4) compared with the 43±12% reduction in the absence of chelerythrine ().

In order to determine whether the effects of PMA on hH1 current were mediated by PKC, additional experiments were performed. First, the effects of the stereoisomer 4-PMA, which does not activate PKC, on hH1 were examined. As shown in Fig 3C, this inactive isomer had no significant effect on hH1 current (mean change of -4±12% at 30 minutes, n=4). If the effects of PMA are mediated by PKC activation, these effects should be blunted or eliminated by inhibition of the kinase. Fig 3D shows averaged data from experiments comparing the effects of PMA with or without preincubation of cells with the PKC inhibitor chelerythrine (20 µmol/L).²⁶ PMA had no significant effect on current (-5±7% after 30 minutes, n=4) when chelerythrine was present.

OAG is a cell membrane–permeant diacylglycerol analogue. Unlike PMA, OAG undergoes hydrolysis after entry into the cell, with transient activation of PKC felt to be more physiological than the persistent activation obtained with phorbol esters. As shown in Fig 4A, OAG also reduced hH1 current amplitude, although the magnitude of this effect was less and the time course somewhat slower than that seen with PMA (22±5% reduction in peak current at 30 minutes, n=3). If suppression of Na⁺ current is in fact mediated through PKC, it should be possible to effect current suppression by activation of an adrenergic receptor that couples to PKC activation in vivo. Therefore, we coexpressed the _1B-adrenergic receptor with hH1 in order to test this hypothesis. In oocytes coexpressing the _1B-receptor and Na⁺ channel, administration of the -adrenergic receptor agonist methoxamine (50 µmol/L) caused a substantial decline in hH1 current (Fig 4B). After 30 minutes of exposure, this reduction averaged 45±10% (n=4). In a separate group of experiments, the effect of methoxamine was completely prevented by chelerythrine preincubation (mean change in current of +6±4%, n=5). Taken together, the experimental results with PMA, OAG, and ₁-receptor stimulation provide compelling evidence that function of the hH1 channel is modulated by activation of PKC with significant suppression of current.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effects of OAG and 1B-receptor stimulation on hH1. A, The diacylglycerol analogue OAG (100 µmol/L) significantly reduced hH1 current (at -10 mV, ) with little effect on cell membrane capacitance (), as shown in this individual experiment. After 30 minutes of exposure, Na+ current declined by 21% in this experiment (mean, 22±5%; n=3). B, After coinjection of hH1 with the 1B-adrenergic receptor, administration of the -agonist methoxamine (50 µmol/L) caused a substantial decline in hH1 current of 51% at 30 minutes in this experiment (mean, 45±10%; n=4).

The consensus phosphorylation site critical for the PKC effect on rBIIA is conserved in hH1 at serine 1503. In order to examine the potential role of this site in the effects of PKC activation on hH1, we mutated this serine to alanine (S1503A) to prevent phosphorylation at this location. Upon expression, Na⁺ currents derived from this mutant channel were similar to wild-type currents with respect to inactivation kinetics and properties of channel gating (data not shown). As shown in Fig 5A, PMA administration suppressed Na⁺ current in the S1503A mutant. However, the reduction in current amplitude was smaller compared with the wild-type channel (see Fig 3B for comparison). In 10 experiments performed with two different mutant recombinants (n=5 each), the decline in I_Na averaged 18±7% at 30 minutes of exposure. The effect of PMA was also more variable in the S1503A mutant, with two experiments showing no effect. As with the wild-type hH1 channel, suppression of Na⁺ current by PMA did not result from changes in the properties of hH1 channel gating (data not shown). Similar results were obtained for the S1503A channel with ₁-receptor stimulation. After receptor coexpression, methoxamine caused a smaller reduction in S1503A current (30±8% at 30 minutes, n=5) than that seen with wild-type current (45±10%). In addition, as with PMA, there was no effect of methoxamine on the mutant channel in one of five experiments. These experimental results indicate that although serine 1503 plays a role in the effects of PKC activation on hH1, it cannot fully account for the changes observed, implying the presence of an additional factor(s).

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of PKC activation on S1503A hH1. A, Averaged normalized values for peak Na+ current derived from expression of the S1503A mutant channel () and capacitance () are shown over time after addition of PMA (10 nmol/L) with a 34±9% reduction in Na+ current at 45 minutes (n=10, *P.05). B, After coinjection of S1503A hH1 with the 1B-adrenergic receptor, administration of the -agonist methoxamine caused a 42% reduction in hH1 current () at 30 minutes in this experiment (mean, 30±8%; n=5).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our experimental results demonstrate that PKC activation reduces Na⁺ current after expression of the human cardiac Na⁺ channel, hH1. This reduction was not associated with any observable change in the voltage dependence or kinetic characteristics of channel gating (although subtle effects cannot be ruled out given the potential limitations of the methods used). Elimination of a conserved consensus PKC site in the interdomain III-IV linker altered the effect of PKC activation but in most cells did not eliminate it. These results demonstrate important differences between hH1 and other Na⁺ channel isoforms in response to stimulation of PKC and suggest that other phosphorylation sites or unknown factors are also involved.

Previous studies have shown that activation of PKC causes a generalized reduction in Na⁺ current in recombinant channels from different tissues and species, although differences exist regarding effects on other properties of channel behavior. Protein kinase modulation of rBIIA has been studied extensively.^{15 16} Like hH1, rBIIA contains multiple consensus sites for phosphorylation by PKC, although most sites are not conserved between the two isoforms. In addition to a reduction in current amplitude, activation of PKC also caused slowing of macroscopic current decay after expression of the rat brain channel in both Xenopus oocytes and a mammalian cell line. In other experiments, a synthetic peptide representing a portion of the rBIIA interdomain III-IV linker containing a putative PKC site (serine 1506) could be rapidly phosphorylated by purified PKC in vitro.¹⁶ Subsequently, when this amino acid was mutated, the functional effects of PKC activation on rBIIA were eliminated. It was concluded that phosphorylation of this single residue (S1506) was the basis of the regulatory effects of PKC on the brain Na⁺ channel.

These findings with rBIIA distinguish it from SkM1 (also known as µ1).²⁷ After expression in a mammalian cell line, activation of PKC led to a marked reduction in SkM1 current that was associated with a negative shift (-15 mV) in the midpoint of the steady state inactivation curve. Interestingly, the rate of macroscopic current decay was faster after PKC activation for this channel. However, when the conserved III-IV linker PKC site in this channel (serine 1321) was mutated, functional modulation by activators of PKC was identical to that of the wild-type channel. Since the interdomain I-II loop is considerably shorter for this channel than for rBIIA and consequently lacks the multiple PKC sites present there in the brain channel, it was proposed that this specific difference in the channel protein structure could be responsible for the S1321-independent regulation by PKC.

Our results indicate that for hH1, the analogous PKC site (S1503) plays at least some role in the response to PKC stimulation, but other factors must also be involved. It is possible that another site on the channel is subject to phosphorylation. A likely candidate region is the interdomain I-II linker, which like rBIIA contains multiple potential PKC sites (and is subject to phosphorylation in the brain channel in that area). Alternatively, other unidentified factors or proteins could also play a role in modulating the PKC effect.

The effects of PKC activation on recombinant rH1 have been previously investigated after expression in two different heterologous expression systems.^{28 29} Using the Xenopus oocyte system, activation of PKC produced effects similar to those seen with hH1, including a reduction in current amplitude without a change in the time course of macroscopic inactivation.²⁹ In addition, a slight shift was observed in the voltage dependence of channel activation to more positive potentials. When rH1 was expressed in a mammalian cell line, activation of PKC not only suppressed Na⁺ current but also was associated with a shift of the steady state inactivation curve in the hyperpolarized direction.²⁸ Thus, the functional effects of PKC activation on the human and rat cardiac isoforms are similar when the two channels are expressed in the same heterologous system (Xenopus oocytes).

Previous studies to investigate the effects of PKC activation on Na⁺ currents in native cardiac myocytes have provided conflicting results. Several groups of investigators have examined the effects of angiotensin II (which activates PKC) on Na⁺ currents in cardiac ventricular cells from different species.^{11 12 13} In two studies,^{11 12} angiotensin II caused an increase in Na⁺ current amplitude in neonatal rat cardiac myocytes. However, the effects of direct PKC activators were not consistent, as a phorbol ester increased current amplitude in one study,¹¹ whereas OAG led to a reduction in peak Na⁺ current in another.¹² In a separate study using isolated guinea pig ventricular myocytes, angiotensin II demonstrated a dose-dependent effect, with an increase in Na⁺ current amplitude at concentrations up to 1 µmol/L but a reduction in current at higher concentrations.¹³ The reasons for these discrepancies are unclear. Potential explanations include differences in recording techniques, experimental temperature, voltage-clamp protocols used, and PKC isoforms present. Finally, it is possible that tissue-specific (cardiac) Na⁺ channel isoforms from different species could respond differently to kinase stimulation. An example of this was demonstrated for the recombinant K⁺ channel protein, min K, which is currently thought to be responsible at least in part for I_Ks in cardiac myocytes.^{30 31} The mouse isoform of min K was not affected by stimulation of PKA, which is known to increase I_Ks in native myocytes.³² However, K⁺ current derived from the guinea pig isoform did increase with administration of a cAMP analogue,³³ suggesting that species differences in the channel subunit amino acid sequence were responsible for the disparate effects. These studies highlight the importance of studying human cardiac Na⁺ channels to understand the functional effects of kinase modulation on Na⁺ current in human heart.

In summary, these results demonstrate that activation of PKC produces functional effects on Na⁺ currents derived from expression of the human cardiac Na⁺ channel hH1, with marked suppression in current amplitude. These effects depend at least in part on the presence of a conserved potential PKC site in the III-IV interdomain linker serine 1503. The functional effects that we observed are similar to those seen with the rat cardiac isoform but appear to differ from rat brain and skeletal muscle channels. These findings indicate that modulation of Na⁺ channel function by PKC is isoform specific, with a molecular basis of effect that differs between channels. It is likely that modulation of hH1 by PKC could have important clinical consequences. In vivo, PKC activation would occur during states of neurohumoral activation, eg, with elevated concentrations of plasma norepinephrine as occurs in congestive heart failure. A PKC-mediated reduction in Na⁺ current could lead to a local slowing of electrical impulse conduction and could thereby potentially facilitate the development of reentrant ventricular tachyarrhythmias. Through this mechanism, the suppression of Na⁺ current mediated by PKC activation could contribute to the high incidence of sudden cardiac death seen in these patients.

	Selected Abbreviations and Acronyms

 

= time constant fast, slow = fast and slow components of hH1 = human cardiac Na+ channel IKs = slowly activating component of delayed rectifier K+ current k = slope factor OAG = 1-oleoyl-2-acetyl-glycerol PKA, PKC = protein kinase A and C PMA = phorbol 12-myristate 13-acetate rBIIA = rat brain IIA Na+ channel rH1 = rat cardiac Na+ channel SkM1 = skeletal muscle Na+ channel V½ = midpoint

	Acknowledgments

 
This study was supported by grants from the US Public Health Service (HL-02363 and HL-46681) and the American Heart Association, Tennessee Affiliate.

	Footnotes

 
Reprint requests to Dr Katherine T. Murray, Room 559, Medical Research Building II, Department of Pharmacology, Vanderbilt University School of Medicine, 21st and Pierce, Nashville, TN 37232-6602. E-mail kathy.murray@mcmail.vanderbilt.edu

Received November 25, 1996; accepted December 27, 1996.

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