Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Shigeki Kobayashi,Mark L Bannister,Jaya P Gangopadhyay,Tomoyo Hamada,Jerome Parness,Noriaki Ikemoto

doi:10.1074/jbc.m408375200

Shigeki Kobayashi, Mark L Bannister + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m408375200

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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 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.

Highlights

Dantrolene (hydrated 1-(((5-(4-nitrophenyl)-2-furanyl)methylene)amino)-2,4-imidazolidinedione sodium salt) is an intracellularly acting skeletal muscle relaxant used for the treatment of malignant hyperthermia (MH).1 MH is a potentially
Since the 51-kDa methylcoumarin acetate (MCA)-labeled region is included in the N-terminal MH domain encompassed by Cys35–Arg614, these results indicate that DP4-mediated MCA labeling has taken place within the presumed N-terminal domain portion of the domain switch
These results suggest that DP4, Leu2442–Pro2477 of the central domain of RyR1, binds to the N-terminal domain, as predicted from our domain switch hypothesis

Summary

Introduction

Dantrolene (hydrated 1-(((5-(4-nitrophenyl)-2-furanyl)methylene)amino)-2,4-imidazolidinedione sodium salt) is an intracellularly acting skeletal muscle relaxant used for the treatment of malignant hyperthermia (MH). MH is a potentially. Dantrolene has been shown to at least partially restore the normal properties of RyR1 Ca2ϩ channels in SR isolated from MH-susceptible pigs [1, 17, 18] These studies together suggest that dantrolene interacts with the RyR to suppress the channel dysfunction that occurs with MH mutations. The Ca2ϩ release properties of expressed RyR1 channels containing randomly selected MH mutations from the N-terminal and central domains have been shown to display similar properties of hyperactivation and hypersensitization [27]. To explain these results, we have proposed a model of channel regulation that involves interdomain interactions between the N-terminal and central domains of RyR1 serving as a “domain switch” for Ca2ϩ channel regulation (28 –31). We present new evidence suggesting that this is at least a portion of the mechanism of action of dantrolene

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