C2 region-derived peptides of β-protein kinase C regulate cardiac Ca²⁺ channels

We have previously shown that α₁-adrenergic activation inhibited β- adrenergic stimulated L-type Ca²⁺ current (1(Ca)). 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 (I(Ba)) was recorded through Ca²⁺ channels in adult rat ventricular myocytes. We found that PMA (0.1 μmol/L) consistently inhibited basal I(Ba) by 40.5 ± 7.4% and isoproterenol (ISO, 0.1 μmol/L)-stimulated I(Ba), 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, β-PKC, and β₁₁-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 I(Ha) 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 I(Ba) 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 BC2-4, PMA-induced inhibition of basal I(Ba) was 26.1 ±4.5% and 23.6±2.2%, respectively. Similarly, ISO-stimulated I(Ba) 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 I(Ba) 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.