Dantrolene stabilizes domain interactions within the ryanodine receptor

Abstract

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca2+ channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (KQ) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 °C and was unaffected by calmodulin. [3H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca2+ release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 °C, yet required the presence of calmodulin at 22 °C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca2+ channel opening rather than the domain switch itself. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.