Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase

We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned card iac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or b eta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and t wo-electrode voltage-clamp recordings were made 2 or 3 days later. Applicat ion of 8-Br-cGMP caused decreases in calcium channel currents in cells expr essing the alpha(1C) subunit, whether or not a beta subunit was co-expresse d. No inhibition of currents by 8-Br-cGMP was observed in the presence of t he protein kinase G inhibitor KT5823. Substitutions of serine residues by a lanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subuni t. As for wild type, the mutant S1371A exhibited inhibition of calcium chan nel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type chan nels but not for the mutant S533A These results indicate that cGMP causes i nhibition of L-type calcium channel currents by phosphorylation of the alph a(1C) subunit at position Ser(533) via the action of protein kinase G.