C2 region-derived peptides of beta-protein kinase C regulate cardiac Ca2+ channels.
We have previously shown that alpha1-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 4beta-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 micromol/L) consistently inhibited basal I(Ba) by 40.5+/-7.4% and isoproterenol (ISO, 0.1 micromol/L)-stimulated I(Ba) by 48.9+/-7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue alpha-phorbol 12,13-didecanoate (0.1 micromol/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 (betaC2-2 and betaC2-4) specifically inhibit the translocation and function of C2-containing isozymes (alpha-PKC, betaI-PKC, and betaII-PKC), but not the C2-less isozymes (delta-PKC and epsilon-PKC). We first used the pseudosubstrate peptide (0.1 micromol/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 micromol/L of betaC2-2 or betaC2-4, 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 betaC2-2 and betaC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled betaC2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated I(Ba) was almost completely abolished in cells dialyzed with both betaC2-2 and betaC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca2+ channel activity.
Zhang
Z H
ZH
Department of Medicine, State University of New York, Health Science Center, and the Veterans Administration Medical Center, Brooklyn 11209, USA.
Johnson
J A
JA
Chen
L
L
El-Sherif
N
N
Mochly-Rosen
D
D
Boutjdir
M
M
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004095:Dihydropyridines / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D011941:Receptors, Adrenergic / Q000502:physiology; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000318:Adrenergic beta-Agonists; D015220:Calcium Channels; D004095:Dihydropyridines; D007527:Isoenzymes; D010455:Peptides; D011941:Receptors, Adrenergic; D011493:Protein Kinase C; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate