Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor)

Metabolically S-35-Iabeled calmodulin (CaM) was used to determine the CaM b inding properties of the cardiac ryanodine receptor (RyR2) and to identify potential channel domains for CaM binding. In addition, regulation of RyR2 by CaM was assessed in [H-3]ryanodine binding and single-channel measuremen ts, Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM mo lecules per RyR2 tetramer in the absence of Ca2+; in the presence of 100 mu M Ca2+, the vesicles bound 7.5 CaM molecules per tetramer, Purified RyR2 bo und approximately four [S-35]CaM molecules per RyR tetramer, both in the pr esence and absence of Ca2+. At least four CaM binding domains were identifi ed in [S-35]CaM overlays of fusion proteins spanning the full-length RyR2. The affinity (but not the stoichiometry) of CaM binding was altered by redo x state as controlled by the presence of either GSH or GSSG, inhibition of RyR2 activity by CaM was influenced by Ca2+ concentration, redox state, and other channel modulators. Parallel experiments with the skeletal muscle is oform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors.