Ca2+/Calmodulin-Dependent Phosphorylation of the Ca2+-ATPase, Uncoupled from Phospholamban, Stimulates Ca2+-Pumping in Native Cardiac Sarcoplasmic Reticulum

Abstract

Recent studies have demonstrated phosphorylation of the cardiac and slow-twitch muscle isoform (SERCA2a) of the sarcoplasmic reticulum (SR) Ca2+-ATPase (at Ser38) by a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase). Analysis of the functional consequence of Ca2+-ATPase phosphorylation in the native SR membranes, however, is complicated by the concurrent phosphorylation of the SR proteins phospholamban (PLN) which stimulates Ca2+sequestration by the Ca2+-ATPase, and the ryanodine receptor-Ca2+release channel (RYR-CRC) which likely augments Ca2+release from the SR. In the present study, we achieved selective phosphorylation of the Ca2+-ATPase by endogenous CaM kinase in isolated rabbit cardiac SR vesicles utilizing a PLN monoclonal antibody (PLN AB) which inhibits PLN phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which inhibits phosphorylation of RYR-CRC. Analysis of the Ca2+concentration-dependence of ATP-energized Ca2+uptake by SR showed that endogenous CaM kinase mediated phosphorylation of the Ca2+-ATPase, in the absence of PLN and/or RYR-CRC phosphorylation, results in a significant increase (∼50-70%) in the Vmaxof Ca2+sequestration without any change in the k0.5for Ca2+activation of the Ca2+transport rate. On the other hand, treatment of SR with PLN AB (which mimics the effect of PLN phosphorylation by uncoupling Ca2+-ATPase from PLN) resulted in ∼2-fold decrease in k0.5for Ca2+without any change in Vmaxof Ca2+sequestration. These findings suggest that, besides PLN phosphorylation, direct phosphorylation of the Ca2+-ATPase by SR-associated CaM kinase serves to enhance the speed of cardiac muscle relaxation.