Multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca(2+)-induced enhancement of the L-type Ca2+ current in rabbit ventricular myocytes.

Abstract

The intracellular mechanism underlying the Ca(2+)-induced enhancement of the L-type Ca2+ current (ICa) was examined in adult rabbit cardiac ventricular myocytes by using patch-clamp methodology. Internal Ca2+ was elevated by flash photolysis of the Ca2+ chelator Nitr 5, and intracellular Ca2+ levels were simultaneously monitored by Fluo 3 fluorescence. Flash photolysis of Nitr 5 produced a rapid (< 1-second) elevation of internal Ca2+, which led to enhancement (39% to 51% above control) of the peak inward Ca2+ current after a delay of 20 to 120 seconds. Internal dialysis of myocytes with synthetic inhibitory peptides derived from the pseudosubstrate (peptide 273-302) and calmodulin binding (peptide 291-317) regions within the regulatory domain of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) blocked enhancement of ICa produced by elevation of internal Ca2+ but not that produced by beta-adrenergic stimulation. These inhibitory peptides also had no effect on the elevation of internal Ca2+ produced by flash photolysis of Nitr 5. A pseudosubstrate inhibitory peptide derived from protein kinase C had no significant effect on Ca(2+)-dependent enhancement of ICa. We conclude that CaM kinase mediates the Ca(2+)-induced enhancement of ICa in mammalian cardiac myocytes by a mechanism likely involving direct phosphorylation of the L-type Ca2+ channel complex or an associated regulatory protein.