title: 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, Ca(2+)-ATPase, and Ca(2+)-release channel (ryanodine receptor). Phosphorylation of phospholamban and Ca(2+)-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 Ca(2+)-release inhibitor, ruthenium red (RR), and the SR Ca(2+)-release activator, ryanodine (at submicromolar concentrations), on CaM kinase-mediated phosphorylation of the Ca(2+)-cycling proteins in rabbit cardiac SR. Incubation of SR with RR (5-30 microM) for 3 min at 37 degrees C resulted in marked (up to 85%) inhibition of Ca2+ channel phosphorylation (50% inhibition with 15 +/- 2 microM RR) by the endogenous membrane-associated CaM kinase. Phosphorylation of the Ca2+ channel by exogenously added multifunctional alpha CaM kinase II was also inhibited similarly by RR. Phosphorylation of the Ca(2+)-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 microM) caused significant stimulation (up to approximately 50%) of Ca2+ channel phosphorylation but had no effect on Ca(2+)-ATPase and phospholamban phosphorylation. These findings suggest that interaction of RR with the ryanodine receptor induces a "nonphosphorylatable state" of the Ca(2+)-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.
authors.affiliation: Department of Physiology, University of Western Ontario, London, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Netticadan
authors.forename: A
authors.initials: A
authors.lastname: Xu
authors.forename: N
authors.initials: N
authors.lastname: Narayanan
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006352:Heart Ventricles; D007700:Kinetics; D009124:Muscle Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012430:Ruthenium Red / Q000494:pharmacology*; D012433:Ryanodine / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D009124:Muscle Proteins; D010750:Phosphoproteins; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D012433:Ryanodine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium