Rabbit Ventricular Myocytes

The intracellular mechanism underlying the Ca2`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 Ic. produced by elevation of internal Ca ` but not that produced by j-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 Ca2'-dependent enhancement of Ica. We conclude that CaM kinase mediates the Ca2'-induced enhancement of I1c in mammalian cardiac myocytes by a mechanism likely involving direct phosphorylation of the L-type Ca2' channel complex or an associated regulatory protein. (Circ Res. 1994;75:854-861.)