PHOSPHORYLATION OF THE PURIFIED CARDIAC RYANODINE RECEPTOR BY EXOGENOUS AND ENDOGENOUS PROTEIN-KINASES

The ryanodine receptor is the main Ca2+-release structure in skeletal and cardiac sarcoplasmic reticulum. In both tissues, phosphorylation o f the ryanodine receptor has been proposed to be involved in the regul ation of Ca2+ release. In the present study, we have examined the abil ity of the purified cardiac ryanodine receptor to serve as a substrate for phosphorylation by exogenously added catalytic subunit of the cyc lic AMP (cAMP)-dependent protein kinase (PK-A), cyclic GMP (cGMP)-depe ndent protein kinase (PK-G), or calmodulin-dependent protein kinase (P K-CaM). A large amount of phosphate incorporation was observed for PK- CaM (938+/-48 pmol of P(i)/mg of purified channel protein), whereas th e level of phosphorylation was considerably lower with PK-A or PK-G (3 45+/-139 and 96+/-6 pmol/mg respectively). In addition, endogenous PK- CaM activity co-migrates with the ryanodine receptor through several s teps of purification, suggesting a strong association of the two prote ins. This endogenous PK-CaM activity is abolished by a PK-CaM-specific synthetic peptide inhibitor. Endogenous cAMP- and cGMP-dependent phos phorylation was not observed in the purified ryanodine-receptor prepar ation. Taken together, these observations imply that PK-CaM is the phy siologically relevant protein kinase, capable of phosphorylating the c hannel protein to a minimum stoichiometry of 2 mol of P(i) per mol of tetramer.