CA-DEPENDENT FACILITATION OF CARDIAC CA CURRENT IS DUE TO CA-CALMODULIN-DEPENDENT PROTEIN-KINASE

Repetitive membrane potential (E(m)) depolarization from -90 to 0 mV in rabbit and ferret ventricular myocytes induces a facilitation or ''staircase'' of Ca current (I-ca), which is Ca (not E(m)) dependent and takes several seconds to accumulate and dissipate. That is, I-ca at the tenth pulse at 1-2 Hz exceeds that at the first pulse (I-10 > I-1). The I-ca staircase was completely abolished by dialysis with either of two inhibitory peptides of Ca-calmodulin-dependent protein kinase (CaMKII) [CaMKII(290-309) and CaMKII(273-302)], implicating this kinase. Inclusion of ATP-gamma S in the patch pipette gradually increased I-ca but also abolished the staircase implicating phosphorylation. KN-62, a nonpeptide CaMKII inhibitor, reversed the I-ca staircase (I-1 > I-10). However, this effect of KN-62 was largely attributed to a slower recovery from inactivation and a gating shift to more negative E(m) (not seen with CaMKII peptides). Similar results were obtained with H-89 and staurosporine (inhibitors of adenosine 3',5'-cyclic monophosphate and phospholipid-/Ca-dependent protein kinase, respectively). The reversal of the I-ca staircase with H-89 and KN-62 could be prevented by more negative interpulse E(m) or elevation of extracellular [Ca] (which could counteract changes in channel gating due to a reduction in internal negative surface potential). That is, these kinase inhibitors might decrease phosphorylation at the inner membrane surface. In similar to 30% of the cells studied with H-89 and staurosporine the characteristic kinetic difference in I-ca inactivation (faster at I-1 than I-10) was also diminished. This might be due to a relatively nonspecific inhibition of the same protein kinase inhibited by the CaMKII peptides. We conclude that the Ca-dependent I-ca facilitation is due to activation of CaMKII and phosphorylatlion of a site on or near the Ca channel. KN-62, H-89, and staurosporine shifted I-ca gating to more negative potentials and slowed recovery from inactivation, effects that could be due to reduction in phosphorylation at the inner membrane surface. Thus the reversal of the I-ca staircase by KN-62, H-89, and staurosporine may not be Ca channel specific.