Ca2+/calmodulin-dependent phosphorylation of the Ca2+- ATPase, uncoupled from phospholamban, stimulates Ca2+-pumping in native cardiac sarcoplasmic reticulum

Recent studies have demonstrated phosphorylation of the cardiac and slow-tw itch muscle isoform (SERCA2a) of the sarcoplasmic reticulum (SR) Ca2+- ATPa se (at Ser(38)) 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 sti mulates Ca2+ sequestration by the Ca2+-ATPase, and the ryanodine receptor-C a2+ release channel (RYR-CRC) which likely augments Ca2+ release from the S R in the present study, we achieved selective phosphorylation of the Ca2+-A TPase by endogenous CaM kinase in isolated rabbit cardiac SR vesicles utili zing a PLN monoclonal antibody (PLN AB) which inhibits PLN phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which inhibits phosphorylati on of RYR-CRC, Analysis of the Ca2+ concentration-dependence of ATP-energiz ed Ca2+ uptake by SR showed that endogenous CaM kinase mediated phosphoryla tion of the Ca2+-ATPase, in the absence of PLN and/or RYR-CRC phosphorylati on, results in a significant increase (similar to 50-70%) in the V-max of C a2+ sequestration without any change in the k(0.5) for Ca2+ activation of t he Ca2+ transport rate. On the other hand, treatment of SR with PLN AB (whi ch mimics the effect of PLN phosphorylation by uncoupling Ca2+-ATPase from PLN) resulted in similar to 2-fold decrease in k(0.5) for Ca2+ without any change in V-max of Ca2+ sequestration. These findings suggest that, besides PLN phosphorylation, direct phosphorylation of the Ca2+-ATPase by SR-assoc iated CaM kinase serves to enhance the speed of cardiac muscle relaxation, (C) 1999 Academic Press.