Dantrolene stabilizes domain interactions within the ryanodine receptor.
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 Ca(2+) 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 (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees 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 Ca(2+) channel opening rather than the domain switch itself.
Kobayashi
Shigeki
S
Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
Bannister
Mark L
ML
Gangopadhyay
Jaya P
JP
Hamada
Tomoyo
T
Parness
Jerome
J
Ikemoto
Noriaki
N
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D003620:Dantrolene / Q000494:pharmacology*; D005456:Fluorescent Dyes; D008305:Malignant Hyperthermia / Q000188:drug therapy; D008861:Microsomes / Q000737:chemistry; D009125:Muscle Relaxants, Central / Q000494:pharmacology*; D018482:Muscle, Skeletal; D010446:Peptide Fragments / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000187:drug effects; D012519:Sarcoplasmic Reticulum / Q000737:chemistry; D013696:Temperature
D002147:Calmodulin; D005456:Fluorescent Dyes; D009125:Muscle Relaxants, Central; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D003620:Dantrolene; D002118:Calcium