cGMP-Dependent Protein Kinase Regulation of the L-Type Ca 2+ Current in Rat Ventricular Myocytes

Abstract

Abstract Regulation of L-type Ca 2+ channel current [I Ca(L) ] by cGMP-dependent protein kinase (PK-G) was investigated in ventricular myocytes from 2- to 21-day-old rats using whole-cell voltage clamp with internal perfusion. I Ca(L) was elicited by a depolarizing pulse to +10 mV from a holding potential of −40 mV. Stimulated I Ca(L) (by 2 μmol/L isoproterenol) was inhibited to the basal level by internal perfusion with 50 nmol/L PK-G (activated by 8Br-cGMP, 0.1 μmol/L). When I Ca(L) was enhanced by Bay K 8644 (1 μmol/L), the enhanced basal I Ca(L) was also reduced by PK-G. Basal I Ca(L) (nonstimulated through the cAMP/cAMP-dependent protein kinase [PK-A] pathway) was also inhibited to various degrees (large, medium, or small) by internal application of PK-G (25 nmol/L). The average inhibition was 42.1% (n=36), and there were no differences in the inhibition during development. The inhibition by PK-G was blocked by the PK-G substrate peptide (cG-PKI, 300 μmol/L) and by heat inactivation of the PK-G. Relatively specific PK-G inhibitors (eg, cG-PKI and H-8) sometimes reversed the inhibition (5 of 25 cells), whereas isoproterenol stimulated I Ca(L) (7 of 8 cells). When a holding potential of −80 mV was used, the inhibition produced by PK-G was much less. The inhibitory effects of PK-G were not mediated by activating phosphodiesterase or protein phosphatase but most likely by a direct phosphorylation of the Ca 2+ channel or associated regulatory protein. The inhibitory effect of PK-G may be explained by a balance between activities of PK-A and PK-G in regulating the slow Ca 2+ channels at two separate sites.