Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

• Published in: The Journal of biological chemistry 2005-02, Vol.280 (8), p.6580-6587
• Main Authors: Kobayashi, Shigeki, Bannister, Mark L., Gangopadhyay, Jaya P., Hamada, Tomoyo, Parness, Jerome, Ikemoto, Noriaki
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calmodulin - pharmacology; Dantrolene - pharmacology; Fluorescent Dyes; Malignant Hyperthermia - drug therapy; Microsomes - chemistry; Muscle Relaxants, Central - pharmacology; Muscle, Skeletal; Peptide Fragments - pharmacology; Protein Structure, Tertiary; Rabbits; Ryanodine - metabolism; Ryanodine Receptor Calcium Release Channel - chemistry; Ryanodine Receptor Calcium Release Channel - drug effects; Sarcoplasmic Reticulum - chemistry; Temperature
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M408375200

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.