C2 Region–Derived Peptides of β-Protein Kinase C Regulate Cardiac Ca 2+ Channels

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Abstract
We have previously shown that α1-adrenergic activation inhibited β-adrenergic–stimulated L-type Ca2+ current (ICa). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4β-phorbol 12-myristate 13-acetate (PMA). To minimize Ca2+-induced Ca2+ inactivation, Ba2+ current (IBa) was recorded through Ca2+ channels in adult rat ventricular myocytes. We found that PMA (0.1 μmol/L) consistently inhibited basal IBa by 40.5±7.4% and isoproterenol (ISO, 0.1 μmol/L)–stimulated IBa by 48.9±7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue α-phorbol 12,13-didecanoate (0.1 μmol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (βC2-2 and βC2-4) specifically inhibit the translocation and function of C2-containing isozymes (α-PKC, βI-PKC, and βII-PKC), but not the C2-less isozymes (δ-PKC and ε-PKC). We first used the pseudosubstrate peptide (0.1 μmol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated IBa was reduced to 16.8±7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated IBa were then determined in the presence of C2-derived peptides or control peptides. When the pipette contained 0.1 μmol/L of βC2-2 or βC2-4, PMA-induced inhibition of basal IBa was 26.1±4.5% and 23.6±2.2%, respectively. Similarly, ISO-stimulated IBa was inhibited by 29.9±6.6% and 29.3±7.8% in the presence of βC2-2 and βC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled βC2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated IBa was almost completely abolished in cells dialyzed with both βC2-2 and βC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.