Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during β-adrenergic stimulation

Abstract

We aimed to define the relative contribution of both PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca2+ influx to the cell by low [Ca]o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [3H]-ryanodine binding and fast Ca2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca2+. Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βAR stimulati