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

We have previously shown that alpha(1)-adrenergic activation inhibited beta-adrenergic-stimulated L-type Ca2+ current (I-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 4 beta-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 mu mol/L) consistently inhibited basal I-Ba by 40.5 +/- 7.4% and isoproterenol (ISO, 0.1 mu mol/L)-stimulated I-Ba by 48.9+/-7.5%. These inhibitory tory effects were not observed with the inactive phorbol ester analogue alpha-phorbol 12,13-didecanoate (0.1 mu mol/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 (beta C2-2 and beta C2-4) specifically inhibit the translocation and function of C2-containing isozymes (alpha-PKC, beta(I)-PKC, and beta(II)-PKC), but not the C2-less isozymes (delta-PKC and epsilon-PKC). We first used the pseudosubstrate peptide (0.1 mu mol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated I-Ba 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 mu mol/L of beta C2-2 or beta C2-4, 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 beta C2-2 and beta C2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled beta C2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated I-Ba was almost completely abolished in cells dialyzed with both beta C2-2 and beta C2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.