Dantrolene Inhibition of Sarcoplasmic Reticulum Ca Release by Direct and Specific Action at Skeletal Muscle Ryanodine Receptors*

The skeletal muscle relaxant dantrolene inhibits the release of Ca from the sarcoplasmic reticulum during excitation-contraction coupling and suppresses the uncontrolled Ca release that underlies the skeletal muscle pharmacogenetic disorder malignant hyperthermia; however, the molecular mechanism by which dantrolene selectively affects skeletal muscle Ca regulation remains to be defined. Here we provide evidence of a high-affinity, monophasic inhibition by dantrolene of ryanodine receptor Ca channel function in isolated sarcoplasmic reticulum vesicles prepared from malignant hyperthermia-susceptible and normal pig skeletal muscle. In media simulating resting myoplasm, dantrolene increased the half-time for Ca release from both malignant hyperthermia and normal vesicles approximately 3.5-fold and inhibited sarcoplasmic reticulum vesicle [H]ryanodine binding (Ki ;150 nM for both malignant hyperthermia and normal). Inhibition of vesicle [H]ryanodine binding by dantrolene was associated with a decrease in the extent of activation by both calmodulin and Ca. Dantrolene also inhibited [H]ryanodine binding to purified skeletal muscle ryanodine receptor protein reconstituted into liposomes. In contrast, cardiac sarcoplasmic reticulum vesicle Ca release and [H]ryanodine binding were unaffected by dantrolene. Together, these results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and Ca. The potential implications of these results for understanding how dantrolene and malignant hyperthermia mutations may affect the voltagedependent activation of Ca release in intact skeletal muscle are discussed.