DIVERGENT EFFECTS OF RUTHENIUM RED AND RYANODINE ON CA2+ CALMODULIN-DEPENDENT PHOSPHORYLATION OF THE CA2+ RELEASE CHANNEL (RYANODINE RECEPTOR) IN CARDIAC SARCOPLASMIC-RETICULUM/

In cardiac muscle, a Ca2+/calmodulin-dependent protein kinase (CaM kin ase) associated with the sarcoplasmic reticulum (SR) is known to phosp horylate the membrane proteins phospholamban, Ca2+-ATPase, and Ca2+-re lease channel (ryanodine receptor). Phosphorylation of phospholamban a nd Ca2+-ATPase is recognized to stimulate Ca2+ sequestration by the SR but the functional consequence of Ca2+ channel phosphorylation has no t been clearly established. In this study, we investigated the effects of the SR Ca2+-release inhibitor, ruthenium red (RR), and the SR Ca2-release activator, ryanodine (at submicromolar concentrations), on Ca M kinase-mediated phosphorylation of the Ca2+-cycling proteins in rabb it cardiac SR. Incubation of SR with RR (5-30 mu M) for 3 min at 37 de grees C resulted in marked (up to 85%) inhibition of Ca2+ channel phos phorylation (50% inhibition with 15 +/- 2 mu M RR) by the endogenous m embrane-associated CaM kinase. Phosphorylation of the Ca2+ channel by exogenously added multifunctional alpha CaM kinase II was also inhibit ed similarly by RR. Phosphorylation of the Ca2+-ATPase by endogenous a nd exogenous CaM kinase was inhibited only modestly (25-30%) by RR, an d phospholamban phosphorylation was unaffected by RR. The magnitude of RR-induced inhibition of Ca2+ channel phosphorylation did not differ appreciably at saturating or subsaturating concentrations of Ca2+ or c almodulin, and in the absence or presence of protein phosphatase inhib itors. In contrast to the effects of RR, low concentrations of ryanodi ne (0.25-1 mu M) caused significant stimulation (up to similar to 50%) of Ca2+ channel phosphorylation but had no effect, on Ca2+-ATPase and phospholamban phosphorylation. These findings suggest that interactio n of RR with the ryanodine receptor induces a ''nonphosphorylatable st ate'' of the Ca2+-release channel, Likely through a conformational cha nge involving occlusion of the CaM kinase phosphorylation site. On the other hand, ryanodine binding to the receptor may serve to maintain a n open, ''phosphorylatable state'' of the channel. (C) 1996 Academic P ress, Inc.