Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation

Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca 2+ and inactivated by higher (∼1 mM) Ca 2+ . To gain insight into a mechanism underlying Ca 2+ -dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [ 3 H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca 2+ -dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2–S3 loop) greatly reduced Ca 2+ -dependent channel inactivation. Activities of these mutant channels were suppressed at 10–100 μM Ca 2+ , and the suppressions were relieved by 1 mM Mg 2+ . The Ca 2+ - and Mg 2+ -dependent regulation of S2–S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4–S5 cytoplasmic loop increased Ca 2+ affinities for channel activation and decreased Ca 2+ affinities for inactivation, but impairment of Ca 2+ -dependent inactivation was not as prominent as those of S2–S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca 2+ -dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca 2+ and Mg 2+ . Four malignant hyperthermia-associated RyR1 mutations in the S2–S3 loop conferred RyR2-type Ca 2+ - and Mg 2+ -dependent channel regulation.