Divergent Effects of Ruthenium Red and Ryanodine on Ca2+/Calmodulin-Dependent Phosphorylation of the Ca2+Release Channel (Ryanodine Receptor) in Cardiac Sarcoplasmic Reticulum

Abstract

In cardiac muscle, a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the sarcoplasmic reticulum (SR) is known to phosphorylate the membrane proteins phospholamban, Ca2+-ATPase, and Ca2+-release channel (ryanodine receptor). Phosphorylation of phospholamban and Ca2+-ATPase is recognized to stimulate Ca2+sequestration by the SR but the functional consequence of Ca2+channel phosphorylation has not 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 CaM kinase-mediated phosphorylation of the Ca2+-cycling proteins in rabbit cardiac SR. Incubation of SR with RR (5–30 μM) for 3 min at 37°C resulted in marked (up to 85%) inhibition of Ca2+channel phosphorylation (50% inhibition with 15 ± 2 μMRR) by the endogenous membrane-associated CaM kinase. Phosphorylation of the Ca2+channel by exogenously added multifunctional α CaM kinase II was also inhibited similarly by RR. Phosphorylation of the Ca2+-ATPase by endogenous and exogenous CaM kinase was inhibited only modestly (25–30%) by RR, and 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 calmodulin, and in the absence or presence of protein phosphatase inhibitors. In contrast to the effects of RR, low concentrations of ryanodine (0.25–1 μM) caused significant stimulation (up to ∼50%) of Ca2+channel phosphorylation but had no effect on Ca2+-ATPase and phospholamban phosphorylation. These findings suggest that interaction of RR with the ryanodine receptor induces a “nonphosphorylatable state” of the Ca2+-release channel, likely through a conformational change involving occlusion of the CaM kinase phosphorylation site. On the other hand, ryanodine binding to the receptor may serve to maintain an open, “phosphorylatable state” of the channel.