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Am J Physiol Cell Physiol 281: C1477-C1486, 2001;
0363-6143/01 $5.00
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Vol. 281, Issue 5, C1477-C1486, November 2001

Evidence for functional role of epsilon PKC isozyme in the regulation of cardiac Na+ channels

Guang-Qian Xiao1, Yongxia Qu1, Zhou-Qian Sun1, Daria Mochly-Rosen2, and Mohamed Boutjdir1

1 Molecular and Cellular Cardiology Program, Veterans Affairs New York Harbor Healthcare System, and State University of New York Health Science Center, Brooklyn, New York 11209; and 2 Department of Molecular Pharmacology, Stanford University, Stanford, California 94305


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Investigation of the role of individual protein kinase C (PKC) isozymes in the regulation of Na+ channels has been largely limited by the lack of isozyme-selective modulators. Here we used a novel peptide-specific activator (epsilon V1-7) of epsilon PKC and other peptide isozyme-specific inhibitors in addition to the general PKC activator phorbol 12-myristate 13-acetate (PMA) to dissect the role of individual PKCs in the regulation of the human cardiac Na+ channel hH1, heterologously expressed in Xenopus oocytes. Peptides were injected individually or in combination into the oocyte. Whole cell Na+ current (INa) was recorded using two-electrode voltage clamp. epsilon V1-7 (100 nM) and PMA (100 nM) inhibited INa by 31 ± 5% and 44 ± 8% (at -20 mV), respectively. These effects were not seen with the scrambled peptide for epsilon V1-7 (100 nM) or the PMA analog 4alpha -phorbol 12,13-didecanoate (100 nM). However, epsilon V1-7- and PMA-induced INa inhibition was abolished by epsilon V1-2, a peptide-specific antagonist of epsilon PKC. Furthermore, PMA-induced INa inhibition was not altered by 100 nM peptide-specific inhibitors for alpha -, beta -, delta -, or eta PKC. PMA and epsilon V1-7 induced translocation of epsilon PKC from soluble to particulate fraction in Xenopus oocytes. This translocation was antagonized by epsilon V1-2. In native rat ventricular myocytes, PMA and epsilon V1-7 also inhibited INa; this inhibition was antagonized by epsilon V1-2. In conclusion, the results provide evidence for selective regulation of cardiac Na+ channels by epsilon PKC isozyme.

protein kinase C; two-electrode voltage clamp; peptides; Xenopus oocyte; electrophysiology


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC Na+ channels determine cell excitability and are responsible for conduction velocity of the action potential. They are the targets of several anti-arrhythmic drugs (13) and kinases (12, 51). Both protein kinase A and protein kinase C (PKC) have been implicated in the modulation of Na+ channels(12, 51). Two subfamilies of PKC isozymes can be stimulated by the tumor-promoting drug 4beta -phorbol 12-myristate 13-acetate (PMA): the conventional PKC (cPKC) isozymes alpha -, beta I-, beta II-, and gamma PKC, which contain the Ca2+ binding domain (C2-containing), and the novel PKC (nPKC) isozymes delta -, theta -, epsilon -, and eta PKC, or C2-less isozymes (8).

The regulation of Na+ channels by PKC has been studied using general PKC activators such as PMA (7) and 1-oleoyl-2-acetyl-sn-glycerol (34). In general, activation of PKC by these non-isozyme-specific activators leads to a reduction in Na+ current (INa) in both brain and heart (25, 31, 32). The characterization of the role of individual PKC isozymes in the regulation of ion channels in general and Na+ channels in particular has been largely limited by the lack of isozyme selective activators and inhibitors. Identification of the particular isozyme(s) that mediates the regulation of Na+ channels is essential for our better understanding of the regulation of INa in physiological and pathological settings. Recently, we have demonstrated, using novel peptide activators and inhibitors of individual isozymes (16, 54), that C2-containing isozymes and epsilon PKC play an important role in mediating PMA-induced inhibition of L-type Ca2+ channels. PKC activation has been associated with the translocation of PKC isozymes from one intracellular compartment to another (10, 27). This translocation event is required for the functional PKC isozymes (40) and is mediated, at least in part, by the binding of activated PKC isozymes to the selective anchoring proteins (RACKs, or receptors for activated C-kinase) that anchor them to different subcellular sites and consequently activate them (28). Anchoring is required for the proper function of individual PKC isozymes. Inhibition or activation of anchoring will alter function. Peptides that mimic either the PKC binding site on RACKs or the RACK binding site on PKC are translocation inhibitors of PKC that inhibit the function of the enzyme (41). On the other hand, a peptide that binds PKC, opens up PKC structure, exposes the catalytic site, and enables anchoring to RACKs will be a PKC agonist (41). On the basis of this rationale, peptide inhibitors and activators of particular PKC isozymes have been developed to inhibit and activate interaction of individual PKC isozymes with their respective RACKs, thus altering their translocation and function as well (19, 41). Using these peptides, we examined the potential role of individual PKC isozymes in the regulation of cloned human Na+ channels expressed in Xenopus oocytes and of Na+ channels in rat ventricular myocytes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of Xenopus oocyte and cRNA injection. Mature female Xenopus frogs, purchased from Xenopus I (Ann Arbor, MI), were anesthetized with 1.5 mg/ml tricaine. Surgically removed ovarian lobes were dissected and treated for 1.5 h with 1.5 mg/ml collagenase type IA dissolved in Ca2+-free ND96 medium (in mM: 96 NaCl, 2 KCl, 2 MgCl2, and 5 HEPES, pH 7.4). Stage IV and V oocytes were selected. Plasmids encoding human cardiac Na+ channel hH1 subunit, pCDNA3.1+-SCN5A, were generously given by Dr. Robert S. Kass (Columbia University, New York, NY). Plasmids were first linearized with restriction enzymes, and in vitro transcription was carried out using the mMSSAGE mMACHINE (Ambion, Austin, TX). Each oocyte was injected with 50 nl of hH1 cRNA. The injected oocytes were stored at 18°C in Leibovitz's L-15 medium (GIBCO BRL, Gaithersburg, MD) supplemented with 50 U/ml penicillin/streptomycin. Currents were recorded from the third to the fourth day.

Isolation of cardiac myocytes. Cardiac myocytes were obtained from hearts of Wistar rats (200-250 g) by enzymatic dissociation as previously described (16, 54). Briefly, hearts were perfused with HEPES-buffered solution containing (in mM) 117 NaCl, 5.4 KCl, 4.4 NaHC03, 1.5 NaH2P04, 1.7 MgCl2, 20 HEPES, 11 glucose, 10 creatine, and 20 taurine. Hearts were then perfused with the same solution containing collagenase type B (1.0-2.0 mg/ml; Boehringer Mannhein, Indianapolis, IN) for 25-30 min. The softened ventricular tissues were removed, cut into small pieces, and mechanically dissociated by trituration. Cells were suspended in petri dishes containing HEPES buffer with 1 mM CaC12 and 0.5% BSA (pH 7.4). All solutions used for perfusion were gassed with 100% O2 and warmed to 37°C. After incubation for 30 min, a small aliquot of the medium containing single cells was transferred to a chamber mounted on the stage of an inverted microscope (Nikon, Tokyo, Japan). Rod-shaped, noncontracting cells with clear striations were used for the whole cell voltage-clamp studies. All experiments were carried out at room temperatures (22-24°C).

Solutions and drugs for oocytes. The composition of external solution for INa recording is ND96 (20). V1- or C2-region-derived peptides (100 nM) were injected individually or in combination, as indicated, in a total volume of 50 nl (1/20 of oocyte volume). Proper diffusion of the peptides into the cytoplasm is reached within 10-15 min as previously reported (48). Ten to fifteen minutes after injection of the antagonist peptide, oocytes were superfused with PMA or 4alpha -phorbol 12,13-didecanoate (4alpha PDD). For epsilon V1-7 (epsilon PKC agonist peptide), the time course of INa was recorded immediately after injection. The peptides epsilon V1-7 [HDAPIGYD; epsilon PKC agonist, also termed pseudo-epsilon RACK (Psi -epsilon RACK)] (10), epsilon V1-2 (EAVSLKPT; epsilon PKC antagonist), alpha C2-4 (SLNPQWNET; alpha PKC antagonist), beta C2-4 (SLNPEWNET; beta PKC antagonist), and eta V1-2 (EAVGLQPT; eta PKC antagonist) were synthesized at Genemed Synthesis (South San Francisco, CA). All peptides used were >90% pure. All chemicals were purchased from Sigma or otherwise indicated.

Solutions and drugs for rat ventricular myocytes. The composition of external solution for INa recordings was (in mM) 100 tetraethylammonium (TEA)-Cl, 15 NaCl, 5 CsCl, 0.1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 adjusted with CsOH). L- and T-type Ca2+ currents were blocked by CoCl2 (5 mM) and NiCl2 (1 mM), respectively. The internal solution contained (in mM) 135 CsOH, 135 L-aspartic acid, 1 MgCl2, 10 EGTA, 10 HEPES, 4 Mg-ATP, and 0.1 Na-GTP (pH 7.1~7.2 adjusted with CsOH). Peptides were added to the peptide solution at a concentration of 100 nM as previously described (16, 54).

Oocyte INa recordings. The expressed INa was recorded with a two-electrode voltage-clamp technique using a GeneCLAMP 500 amplifier (Axon Instrument, Foster City, CA). The volume of the recording chamber was 0.3 ml, and the rate of perfusion was 0.3 ml/min. Oocytes were impaled with electrodes filled with 3 M KCl in ND96 external solution. Oocytes with membrane potential more negative than -40 mV were used for current recording. For INa (20) current-voltage (I-V) relations, oocytes were depolarized from a holding potential of -130 mV to tests ranging from -100 to 70 mV with increments of 5 mV. A depolarization pulse to -20 mV from a holding potential of -130 mV recorded the time course for INa. Oocyte membrane capacitance was calculated from the capacitance transient during a voltage step from -130 to -120 mV. The steady-state inactivation of INa was obtained by using the double-pulse protocol. Prepulse potentials ranging from -130 to 70 mV were applied and then followed by a 5-ms interpulse interval at a potential of -130 mV. The membrane was then depolarized for 200 ms to test potentials of -20 mV. Steady-state inactivation was measured as the ratio of I to Imax (I/Imax), where Imax is the maximum current amplitude elicited during the test pulse to -20 mV after the most hyperpolarizing prepulse. The current ratio was plotted as a function of the prepulse potential. The curves were obtained by fitting the data points with Boltzmann distribution of the form finf(V) = 1/{1 + exp[(Vm - V0.5)/k]}, where finf(V) is the steady-state inactivation parameter, Vm is membrane voltage, V0.5 is the half-maximum inactivation potential, and k is the slope factor. Current recording was done at room temperature (22 ± 2°C).

Myocyte INa recordings. Whole cell INa recording was performed using an Axopatch 200B amplifier with a CV-203BU head stage and pCLAMP software (Axon Instruments). Suction pipettes were made from borosilicate glass capillaries using a horizontal puller (Sutter Instrument, Novato, CA). When filled with pipette solution, tips had resistances ranging between 0.8 and 1.2 MOmega . The tip potential was compensated before the formation of membrane seals. After a seal formed, transient application of negative pressure ruptured the membrane. Hyperpolarizing voltage-clamp steps (to -10 mV from a holding potential of 0 mV) were used to record cell capacitance, which was calculated by integrating the area under the uncompensated capacitance transient and dividing this area by the voltage step. Cell capacitance and pipette series resistance were both compensated before the onset of the experiment. To record INa time course, cells were depolarized to -25 mV for 50 ms from a holding potential of -90 mV (24). All experiments were performed at room temperature (22~24°C). Data acquisition, voltage protocols, and analysis were performed using the pCLAMP suite of software (Axon Instruments,). We allowed 5-8 min for INa to reach steady state and also for peptides to properly enter the cell (16, 54). Therefore, the time 0 shown in Fig. 8 represents about 5-8 min after formation of whole cell configuration.

Immunoprecipitation and Western blot. Stage IV-V oocytes were treated with either 1) PMA (100 nM) or epsilon V1-7 (100 nM) or 2) PMA or epsilon V1-7 plus epsilon V1-2 (100 nM). Membranes were obtained from these oocytes 30 min after the above treatment and purified as previously described (52). Briefly, 50-90 treated oocytes were homogenized in 10% sucrose, 15 mM NaCl, 5 mM KCl, and 20 mM HEPES, pH 7.5, supplemented with proteinase inhibitor cocktail (37). After centrifugation, membrane fractions from 20-50% sucrose gradient interface were collected as particulate fractions, and pellet fractions from 10% and 10-20% sucrose gradient interface were collected as cytosolic fractions. Each fraction was homogenized and solubilized in 2.5 ml of buffer (75 mM KCl, 75 mM NaCl, and 50 mM Na-phosphate, pH 7.2, plus 2 mg/ml soybean lipids and 1% Triton X-100) and centrifuged for supernatant collection.

Mouse-raised antibodies against epsilon PKC (DB Transduction, Piscataway, NJ) were used to immunoprecipitate epsilon PKC. Briefly, anti-epsilon PKC antibody was added to the supernatant, which was precleared with protein A-Sepharose and shaken at 4°C for 4 h. We added 25 µl of 50% protein A-Sepharose beads for every 1 ml of sample and incubated overnight. Protein A-Sepharose antibody/antigen complex was collected by centrifugation, washed, and eluted in reducing SDS sample buffer by boiling for 5 min. For Western blot assay, 35 µl/lane of the above immunoprecipitated proteins were subjected to 8% SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane by electrophoresis. The blot was blocked for 2 h in blocking buffer [5% nonfat dry milk in wash buffer (10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20)] and washed twice in wash buffer. For immunoreaction, the blot was incubated with anti-epsilon PKC antibody at room temperature for 1.5 h. Blots were washed completely with wash buffer. Immunodetection was carried out with a 1:1,500-diluted horseradish peroxidase conjugated anti-mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ) secondary antibody for 1 h at room temperature. Blots were washed again and then incubated with the enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech) for 1 min and exposed to X-ray film.

Data analysis. Data acquired were stored and then analyzed off-line with pCLAMP 6 software (Axon Instruments). All values were measured as the difference between zero and the peak current. All measurements of INa changes were performed at 30 min to avoid potential time-dependent internalization of plasma membrane in oocytes reported after 30 min of exposure to phorbol esters (46). Microcal Origin v5.0 (Microcal Software) was used to generate figures and perform statistical analysis. Data are presented as means ± SE. Percent inhibition was calculated as the difference in the current amplitude caused by the intervention(s), divided by the control value. Student's paired t-test was used to compare the data before and after interventions. Unpaired t-test or ANOVA was used to compare the data between groups. A value of P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PMA inhibited INa expressed in Xenopus oocytes. To investigate whether, under our experimental conditions, PKC is involved in the modulation of INa, we first used a general PKC activator, PMA, and a general PKC inhibitor, calphostin C. Figure 1 shows the effect of PMA in the absence (Fig. 1, A and B) and presence of calphostin C (Fig. 1D). Exposure of oocytes to PMA (100 nM) resulted in a slow and time-dependent inhibition of peak INa (Fig. 1A). The I-V relations during control and PMA at 30 min are shown in Fig. 1B. PMA inhibited INa by 44.3 ± 8.2% at -20 mV (n = 8, P < 0.05 compared with control). The specificity of PMA effects on INa was confirmed by comparing its effects to another phorbol ester, 4alpha PDD, which does not activate PKC (11, 44). PMA effects were not seen [2.9 ± 2.2%, n = 5, P = not significant (NS) compared with control] with its inactive analog, 4alpha PDD, at the same concentration of 100 nM (Fig. 1C). PMA and 4alpha PDD effects at 30 min on the cell capacitance of oocytes are shown in the lower part of Fig. 1, A and C, respectively. PMA and 4alpha PDD reduced oocyte cell capacitance by 17 ± 3.7% (n = 6, P < 0.05) and 15 ± 3.6% (n = 5, P < 0.05), respectively. Calphostin C superfusion for 10-15 min before the onset of PMA application completely blocked PMA inhibition of INa (only 4.6 ± 3.3%, n = 4, P = NS compared with control). These experiments indicate that PMA inhibition of INa is mediated through PKC.


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  		Fig. 1.   Inhibition of Na+ current (INa) by phorbol 12-myristate 13-acetate (PMA). A: time course of peak INa inhibition by PMA (100 nM). B: current-voltage (I-V) relations of INa during control and 30 min after superfusion of PMA (100 nM) in 6 oocytes. C: specificity of PMA effects on INa as confirmed by comparing its effects to another phorbol ester, 4alpha -phorbol 12,13-didecanoate (alpha PDD; 100 nM), which does not activate protein kinase C (PKC). Oocyte capacitance expressed in fractional values is shown in A and C (bottom). D: I-V relations of INa during control (n = 4) and 30 min after PMA superfusion of 4 oocytes pretreated with calphostin C, a general PKC inhibitor. Selected INa tracings (-20 mV) at the times indicated by a and b are shown in insets in A and C. Time 0 corresponds to the time of oocyte impalement; arrows refer to the onset of drug superfusion.

Peptide activator of epsilon PKC, epsilon V1-7, inhibited INa expressed in Xenopus oocytes. To selectively activate epsilon PKC, we used a novel peptide, epsilon V1-7, also termed pseudo-epsilon RACK (Psi -epsilon RACK), which is derived from the regulatory V1 region of epsilon PKC and was previously shown to selectively activate the translocation of epsilon PKC (10). This is the first and only available agonist peptide activator of one single PKC isozyme. Figure 2 shows the effect of this epsilon PKC agonist, epsilon V1-7, and its scrambled peptide (negative control) on INa. Recording of INa began immediately after injection of epsilon V1-7 (100 nM) or its scrambled peptide (100 nM). Figure 2A illustrates the time course of epsilon V1-7 inhibition of INa from one oocyte. epsilon V1-7 at 30 min reduced oocyte cell capacitance by 3.9 ± 3.0% (Fig. 2A, bottom; n = 5, P = NS). Figure 2B shows the I-V relations of INa during control and 30 min after the injection of epsilon V1-7. epsilon V1-7 inhibited peak INa by 30.5 ± 4.5% (n = 15, P < 0.05 compared with control). Figure 2C shows the lack of effect of epsilon V1-7 scrambled peptide on INa I-V relations (3.2 ± 2%, n = 5, P = NS compared with control). However, PMA superfusion of seven other oocytes preinjected with epsilon V1-7 scrambled peptide resulted in INa inhibition by 40.1 ± 6.3% (n = 7, P < 0.05 compared with epsilon V1-7 scrambled peptide).


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  		Fig. 2.   Inhibition of INa by peptide epsilon V1-7. A: time course of peak INa recorded in an oocyte injected with the peptide epsilon V1-7 (100 nM), a specific epsilon PKC activator. Bottom: oocyte capacitance expressed in fractional values. Inset: selected INa tracings (-20 mV) at the times indicated by a (control) and b (30-min perfusion of PMA). B: I-V relations of INa during control and 30 min after injection of epsilon V1-7 in 8 oocytes. C: I-V relations of INa during control (n = 5) and 30 min after injection of epsilon V1-7 scrambled peptide, followed by 30 min of superfusion of PMA (100 nM) in the same oocytes injected with epsilon V1-7 scrambled peptide (n = 5).

To further evaluate the selectivity of the peptide epsilon V1-7 on INa and its mechanism in the regulation of INa, we studied the effect of epsilon V1-7 on INa in the presence of a peptide-specific inhibitor of epsilon PKC, epsilon V1-2, which has been shown to selectively inhibit the translocation of epsilon PKC (10). Figure 3 shows the effect of PMA (100 nM) and epsilon V1-7 (100 nM) on INa in the presence of epsilon V1-2 peptide (100 nM). Figure 3A shows the time course of the PMA effect on peak INa from one oocyte injected with epsilon V1-2. Figure 3B shows the I-V relations during control and 30 min after PMA application in oocytes injected with epsilon V1-2 peptide. epsilon V1-2 peptide antagonized the PMA inhibitory effect on INa (only 8.1 ± 3.5%, n = 5, P < 0.05 compared with PMA alone). Figure 3C shows the time course of peak INa from one oocyte after the coinjection of both epsilon V1-7 and epsilon V1-2 peptides. epsilon V1-7 failed to significantly inhibit INa in the presence of epsilon V1-2, indicating that epsilon V1-7 inhibited INa by functionally activating the translocation of epsilon PKC. Figure 3D shows the I-V relations during control and 30 min after the coinjection of epsilon V1-2 plus epsilon V1-7. The effect of epsilon V1-7 on INa was completely blocked by epsilon V1-2 peptide (4.4 ± 3.1%, n = 6, P = NS compared with 4alpha PDD). Together, these results demonstrate the ability of the novel peptide epsilon V1-7 to activate one single PKC isozyme, epsilon PKC, and the ability of peptide epsilon V1-2 to block these effects, thus altering INa channel function.


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  		Fig. 3.   Effect of PMA and epsilon V1-7 peptide on INa in the presence of the peptide inhibitor of epsilon PKC, epsilon V1-2. A: time course of peak INa recorded from an oocyte injected with epsilon V1-2 (100 nM) and superfused with PMA (100 nM). B: I-V relations of INa during control and 30 min after superfusion of PMA in 5 oocytes injected with epsilon V1-2 peptide. C: time course of peak INa recorded from an oocyte injected with both epsilon V1-2 (100 nM) and epsilon V1-7 (100 nM) peptides. D: I-V relations during control and 30 min after injection of both epsilon V1-7 (100 nM) and epsilon V1-2 (100 nM) peptides in 5 oocytes. Selected INa tracings (-20 mV) at the times indicated are shown in insets in A and C. Time 0 corresponds to the time of oocyte impalement; arrows refer to the onset of drug superfusion.

PMA inhibition of INa was not altered by peptide-specific antagonists of alpha -, beta -, delta -, and eta PKC isozymes. Figure 4A shows the I-V relations of INa during control and 30 min after PMA superfusion of oocytes injected with alpha C2-4 peptide; INa was decreased by 44.9 ± 7.9% (n = 6, P = NS compared with PMA alone). Figure 4B shows the I-V relations of INa during control and 30 min after PMA superfusion of oocytes injected with beta C2-4 peptide; INa was decreased by 43.6 ± 6.9% (n = 5, P = NS compared with PMA alone). Figure 4C shows the I-V relations of INa during control and 30 min after PMA superfusion of oocytes injected with delta V1-1 peptide; INa was decreased by 44.5 ± 8.2% (n = 5, P = NS compared with PMA alone). Figure 4D shows the I-V relations of INa during control and 30 min after PMA superfusion of oocytes injected with eta V1-2 peptide; INa was decreased by 43.9 ± 7.0% (n = 7, P = NS, compared with PMA alone). These results demonstrate that alpha -, beta -, delta -, and eta PKC isozymes did not alter PMA-induced INa inhibition. A summary of all the above results is shown in Fig. 5.


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  		Fig. 4.   Effect of PMA on INa in the presence of peptide inhibitors of alpha -, beta -, delta -, and eta PKC isozymes. A-D: I-V relations of INa during control and 30 min after superfusion of PMA (100 nM) in the presence of alpha C2-4 (100 nM, n = 6), beta C2-4 (100 nM, n = 5), delta V1-1 (100 nM, n = 5), and eta V1-2 (100 nM, n = 7) peptides, respectively.



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  		Fig. 5.   Summary of percent inhibition of INa by PMA, alpha PDD, calphostin C, and currently available PKC isozyme-specific peptide agonist and antagonists. *P value indicates comparison with PMA alone. **P value indicates comparison with alpha PDD superfusion. ***P value indicates comparison with epsilon V1-7 alone. NS, not significant.

PMA and epsilon V1-7 did not alter steady-state inactivation of INa expressed in Xenopus oocytes. The effects of PMA and epsilon V1-7 on steady-state inactivation of INa were also investigated using the double-pulse protocol as indicated in MATERIALS AND METHODS. Figure 6 shows the averaged normalized data plotted against the prepulse potentials for PMA (A) and epsilon V1-7 (B). The curves in Fig. 6 were obtained by fitting the data points with the Boltzmann distribution described in MATERIALS AND METHODS. The inactivation curves were nearly identical between control and either PMA or epsilon V1-7 in Fig. 6, A or B, respectively, suggesting that PMA or peptide epsilon V1-7 did not change the kinetics of voltage-dependent inactivation of INa. For the control group (n = 6), V0.5 was -80 ± 4.3 mV and k was 4.4 ± 0.7 mV, whereas for the PMA group (n = 6), V0.5 was -84 ± 6.8 mV and k was 4.3 ± 0.9 mV. Similarly, for the control group (n = 5), V0.5 was -79.7 ± 3.8 mV and k was 4.5 ± 0.5 mV, whereas for the epsilon V1-7 group (n = 5), V0.5 was -82.6 ± 6.4 mV and k was 4.4 ± 0.8 mV.


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  		Fig. 6.   Effect of PMA and epsilon V1-7 on INa steady-state inactivation curves. Steady-state inactivation curves were fit through mean data points using the Boltzmann equation finf(V) = 1/{1 + exp[(Vm - V0.5)/k]}, where Vm is membrane voltage, V0.5 is the half-maximum inactivation potential, and k is the slope factor. A: V0.5 was -80 ± 4.3 mV and k was 4.4 ± 0.7 mV for control (n = 6), and V0.5 was -84 ± 6.8 mV and k was 4.3 ± 0.9 mV for PMA (n = 6). B: V0.5 was -79.7 ± 3.8 mV and k was 4.5 ± 0.5 mV for control (n = 5), and V0.5 was -82.6 ± 6.4 mV and k was 4.4 ± 0.8 mV for epsilon V1-7 (n = 5).

PMA and epsilon V1-7 induced translocation of epsilon PKC in Xenopus oocytes. To demonstrate that the functional inhibition of INa is associated with biochemical translocation of epsilon PKC from the cytosol to the membrane, we performed Western blot assays on oocytes treated with epsilon PKC activators and/or inhibitors. Figure 7 shows the translocation of epsilon PKC in oocytes by PMA (100 nM) and epsilon V1-7 (100 nM) and its inhibition by epsilon V1-2 (100 nM). Figure 7, lanes 2 and 4, show that epsilon PKC was translocated from the soluble to the particulate fraction by PMA and epsilon V1-7, respectively. Figure 7, lanes 6 and 8, show that epsilon V1-2 antagonized PMA- and epsilon V1-7-induced epsilon PKC translocation from the soluble to the particulate fraction, respectively. Similar results were obtained in a total of four independent experiments.


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  		Fig. 7.   Translocation of epsilon PKC by PMA and epsilon V1-7: Western blot analysis was performed in oocytes treated with PMA (100 nM) and/or epsilon PKC peptide modulators (100 nM). Lanes 2 and 4 show that epsilon PKC translocated from the soluble (S) to the particulate (P) fractions by PMA and epsilon V1-7, respectively. Lanes 6 and 8 show that epsilon V1-2 antagonized PMA- and epsilon V1-7-induced epsilon PKC translocation from soluble to particulate fractions, respectively.

PMA and epsilon V1-7 inhibited INa in rat ventricular myocytes. We next tested whether epsilon PKC also modulates INa in native cardiac myocytes. INa was recorded from rat ventricular cells, and the effects of epsilon PKC activation were investigated. Figure 8A shows the time course of the effect of 100 nM PMA on INa in the absence or presence of intrapipette epsilon V1-2 (100 nM). PMA inhibited INa by 53 ± 5.9% (n = 5, P < 0.05). However, in the presence of epsilon V1-2, PMA-induced INa inhibition was reduced to 22.4 ± 7% (n = 4, P < 0.05 compared with PMA alone). Figure 8B shows the time course of the effect of epsilon V1-7 (100 nM) on INa in the absence and presence of intrapipette epsilon V1-2 (100 nM). epsilon V1-7 inhibited INa by 34.3 ± 5.4% (n = 6, P < 0.05). This inhibition of INa by epsilon V1-7 was completely abolished by epsilon V1-2 (5.4 ± 1.4%, n = 4, P > 0.05). All together, the data obtained in native cardiac myocytes indicate that selective epsilon PKC activation also inhibited INa. These results are similar to those obtained in Xenopus oocytes.


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  		Fig. 8.   Effects of PMA and epsilon V1-7 on INa in rat ventricular myocytes. A: time course of peak INa from 2 different myocytes superfused with PMA (100 nM, open circle ) and PMA + epsilon V1-2 (100 nM, ). B: time course of peak INa from 2 different myocytes dialyzed with epsilon V1-7 (100 nM, open circle ) and epsilon V1-2 (100 nM) + epsilon V1-7 (). Insets: selected current traces at the times indicated by a and b.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study is the first to show that epsilon PKC is involved in PMA-induced inhibition of the cloned human INa expressed in Xenopus oocyte and INa recorded from rat ventricular myocytes. It is evident that identification of the particular isozyme(s) that mediates the regulation of Na+ channels is of important therapeutic implications.

Regulation of INa by PKC. The characterization of the role of individual PKC isozymes in the regulation of ion channels in general and Na+ channels in particular has been largely limited by the lack of isozyme selective activators and inhibitors. While several previous studies implicated PKC in the regulation of Na+ channels, the role and the identity of the isozyme(s) responsible for this regulation remain largely unexplored. Heterologously expressed rat brain (rBIIA) (7, 32) and human cardiac Na+ channel currents (hH1) (31) were reduced upon PKC activation. Although both rBIIA and hH1 contain consensus sites for phosphorylation by PKC, most of the sites are not conserved between these two isoforms. In one study (31), elimination of conserved consensus PKC sites in the hH1 interdomain III-IV linker, which contains the putative PKC site (Ser-1503), does not completely eliminate the PMA-induced INa inhibition, implying that other phosphorylation site(s) may exist.

Our present findings in Xenopus oocytes showing that INa is inhibited by PMA, a general activator of PKC, are consistent with previous studies using a heterologous expression system (7, 31, 34, 35). In addition, the use of a novel peptide-specific activator of epsilon PKC, epsilon V1-7, mimicked PMA effects on INa, and the use of a peptide-specific inhibitor of epsilon PKC, epsilon V1-2, prevented these effects, thus establishing the involvement of epsilon PKC in the regulation of Na+ channels. Although PMA inhibition of INa (44.3%) is slightly higher than epsilon V1-7 inhibition (30.5%), it appears that activation of epsilon PKC alone by the peptide epsilon V1-7 is sufficient to inhibit INa. Because of the unavailability of peptide-specific activators of other PKC isozymes, we do not exclude the possibility that other isoforms may be involved in the regulation of Na+ channels. PMA has been reported to cause time-dependent internalization of plasma membrane in oocytes (46) after 30 min of exposure. In the present study, whereas PMA (100 nM) and 4alpha PDD (100 nM) at 30 min significantly reduced oocyte cell capacitance by about 17% and 15%, respectively, epsilon V1-7 did not significantly alter the oocyte cell capacitance (4%). This finding indicates that PMA effects on oocyte cell capacitance are likely due to a nonspecific effect. Phorbol esters have been reported to mediate some of their responses through beta -chimerin, a member of the GTPase-activating proteins that lacks the functional kinase domain (3, 4). It is therefore possible that some of PMA-nonspecific effects may be mediated through the chimerin family via a yet unknown mechanism. However, PMA inhibition of INa (44.3%) far exceeds the nonspecific effect, suggesting that PMA regulates Na+ channels through a PKC pathway. This is further supported by the fact that part of the PMA effects on INa were reversed by the general PKC antagonist calphostin C and by the epsilon PKC-specific peptide antagonist epsilon V1-2. In addition, biochemical data showed that both PMA and epsilon V1-7 induced the translocation of epsilon PKC from the cytosol to the membrane. This translocation was inhibited by the epsilon PKC-specific peptide inhibitor epsilon V1-2. In the present study, we showed by Western blot that, in Xenopus oocytes, epsilon PKC can be detected, activated, and translocated. This biochemical finding is consistent with our functional data demonstrating that epsilon PKC activation leads to INa inhibition. It is noteworthy that previous studies using Xenopus oocytes have demonstrated the existence of at least six other PKC isozymes including alpha -, beta I-, beta II-, gamma -, delta -, and zeta PKC (9, 18). Similarly, in native rat cardiac myocytes, we showed that epsilon PKC activation results in INa inhibition, consistent with the results obtained in Xenopus and consistent with previously published reports in native cardiac myocytes (34, 49, 50). However, in one study (29), activation of PKC increased INa.

Proposed mechanism of peptide epsilon V1-7 action. Peptide epsilon V1-7 is the first isozyme-selective PKC activator that induces epsilon PKC translocation from the cytosol to the particulate fraction (10). The molecular basis underlying the action of the peptide epsilon V1-7 on epsilon PKC has not been fully explored. It has been suggested that this peptide acts by interfering with the intramolecular interaction within epsilon PKC between the RACK-binding site and the pseudo-RACK site, thereby mimicking the conformational change and dissociation of this intramolecular interaction that occurs upon activation of epsilon PKC, rendering PKC more accessible to its anchoring protein (10). The evidence that a peptide translocation activator for epsilon PKC, epsilon V1-7, functionally inhibited INa suggests that translocation activators should be agonists of PKC function, independent of the amount of second messengers that normally activate PKC. This finding further suggests that the translocation of PKC isozymes is essential for the full function of endogenous PKC activation. Phosphorylation of ion channel proteins is the key mechanism in signal transduction pathways that alter channel properties and influence excitability, and thus the physiological function, of excitable cells (22). The molecular mechanisms by which PKC regulates cardiac Na+ channels are not completely defined.

Physiological and pathophysiological significance of the regulation of INa channels by PKC. In the last few years, research in the general area of signal transduction has advanced significantly. As a result, PKC has emerged as a key component along signal transduction pathways. PKC has been involved in the modulation of ion channels (11, 21, 33, 45, 47, 54), inotropic and chronotropic effects (5, 11, 23, 26, 53), gene expression (6, 38), secretion of cardiac factors (17, 36), hypertrophy (14, 39), ischemia, and infarction (43). It therefore becomes critical to characterize and gain insight on how PKC and, most importantly, its multiple isozymes regulate cardiac ion channels, in both physiological and pathological settings. In the heart, Na+ channels determine excitability and conduction velocity of the action potential (1, 2, 15, 30, 42) and, thus, constitute the key elements in the genesis of arrhythmias. The ability to dissect the individual role of PKC isozymes in the regulation of Na+ channels may provide functional information that will help in the design of isozyme-targeted therapeutics.


    ACKNOWLEDGEMENTS

This study was supported by Veterans Administration Medical Research Funds Merit Grant and REAP Grant (to M. Boutjdir) and by National Heart, Lung, and Blood Institute Grants HL-55401 (to M. Boutjdir) and HL-52141 (to D. Mochly-Rosen).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Boutjdir, Research and Development Office (151), Veterans Affairs New York Harbor Healthcare System, 800 Poly Place, Brooklyn, NY 11209 (E-mail: mohamed.boutjdir{at}med.va.gov).

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. Section 1734 solely to indicate this fact.

Received 14 December 2000; accepted in final form 19 June 2001.


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