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

Metabolically S-35-Iabeled calmodulin (CaM) was used to determine the CaM binding 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 measurements, Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM molecules per RyR2 tetramer in the absence of Ca2+; in the presence of 100 muM Ca2+, the vesicles bound 7.5 CaM molecules per tetramer, Purified RyR2 bound approximately four [S-35]CaM molecules per RyR tetramer, both in the presence and absence of Ca2+. At least four CaM binding domains were identified 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 redox 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 isoform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors.