Characteristics of irreversible ATP activation suggest that native skeletal ryanodine receptors can be phosphorylated via an endogenous CaMKII

Phosphorylation of skeletal muscle ryanodine receptor (RyR) calcium release channels by endogenous kinases incorporated into lipid bilayers with nativ e sarcoplasmic reticulum vesicles was investigated during exposure to 2 mM cytoplasmic ATP. Activation of RyRs after 1-min exposure to ATP was reversi ble upon ATP washout. In contrast, activation after 5 to 8 min was largely irreversible: the small fall in activity with washout was significantly les s than that after brief ATP exposure. The irreversible activation was reduc ed by acid phosphatase and was not seen after exposure to nonhydrolyzable A TP analogs. The data suggested that the channel complex was phosphorylated after addition of ATP and that phosphorylation reduced the RyR's sensitivit y to ATP, adenosine, and Ca 2. The endogenous kinase was likely to be a cal cium calmodulin kinase II (CaMKII) because the CaMKII inhibitor KN-93 and a n inhibitory peptide for CaMKII prevented the phosphorylation-induced irrev ersible activation. In contrast, phosphorylation effects remained unchanged with inhibitory peptides for protein kinase C and A. The presence of CaMKI I beta in the SIR vesicles was confirmed by immunoblotting. The results sug gest that CaMKII is anchored to skeletal muscle RyRs and that phosphorylati on by this kinase alters the enhancement of channel activity by ATP and Ca2 +.