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 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 SternVolmer 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°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°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 Ca 2؉ channel opening rather than the domain switch itself.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 deadly, pharmacogenetically mediated, hypermetabolic response to volatile anesthetics that results from unregulated intramyoplasmic Ca 2ϩ release (1). The drug is known to inhibit excitation-contraction coupling of skeletal muscle (2) by suppressing depolarization-induced sarcoplasmic reticulum (SR) Ca 2ϩ release in normal and MH-susceptible skeletal muscle without affecting voltage sensor activation (3). In MH, the voltage dependence of contractile activation is shifted to lower voltages (4), whereas in the presence of clinical concentrations of dantrolene, i.e. 10 M (5), the voltage dependence of contractile activation is shifted to higher voltages (6, 7). Normalization of the voltage dependence of contractile activation may therefore be one of the important components of the therapeutic action of dantrolene. Dantrolene also confers a normal Mg 2ϩ sensitivity to MH-susceptible muscle fibers, which would otherwise show a considerably reduced sensitivity to the normal inhibitory action of myoplasmic Mg 2ϩ on the SR Ca 2ϩ release mechanism (...

Azumolene Inhibits a Component of Store-operated Calcium Entry Coupled to the Skeletal Muscle Ryanodine Receptor

“…Fourth, we show that substantial inhibition of RyR1-coupled SOCE by azumolene occurs only when cells are treated with this drug prior to C/R-induced RyR1 activation and SR Ca 2ϩ depletion. Because C/R treatment produces prolonged RyR1 channel opening, the inability of azumolene to inhibit SOCE when added after C/R treatment is consistent with previously published in vitro studies demonstrating that dantrolene interacts preferentially with the closed state of RyR1 (36,37). Therefore, we hypothesize that the inhibitory effect of azumolene and, by extension, of dantrolene on SOCE results from drug binding to the closed state of RyR1 The discordance between the ability of azumolene to inhibit SOCE versus SR Ca 2ϩ release in FDB muscle fibers suggests that Ca 2ϩ itself is not the direct signal from RyR1 that stimulates SOCE.…”

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

“…Dantrolene is a hydantoin derivative that is clinically approved for the treatment of Malignant Hyperthermia, a life-threatening skeletal muscle disorder caused by a surge of SR Ca 2ϩ release and Ca 2ϩ entry upon exposure of mutant RyR1 to halothane or other volatile anesthetics (23). Dantrolene binds to a specific domain on RyR1 and has been shown to stabilize proteinprotein interactions supported by this domain (26,37). However, it is unclear whether dantrolene directly inhibits RyR1 opening (54).…”

Dantrolene, a Therapeutic Agent for Malignant Hyperthermia, Inhibits Catecholaminergic Polymorphic Ventricular Tachycardia in a RyR2R2474S/+ Knock-In Mouse Model

“…2- 16 We reported that dantrolene, a specific agent for the treatment of MH, prevented abnormal Ca 2+ leak by correction of the defective inter-domain interaction between the N-terminal and central domains within MH RyR1 (i.e., aberrant formation of a channel-activating unzipped configuration of the N-terminal/central domain pair in an otherwise resting state). 9 We further showed that, in failing hearts, dantrolene corrected the defective inter-domain interaction within the RyR2, thereby inhibiting Ca 2+ leak through RyR2. 11 More recently, by using the knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we clarified that a single amino acid mutation within the RyR2 sensitizes the RyR2 channel to activation by luminal [Ca 2+ ] (i.e., a decreased threshold of luminal [Ca 2+ ] for channel activation), and in turn induces spontaneous Ca 2+ sparks and DAD, leading to CPVT, and that danrrolene stabilized the leaky RyR2 by correcting the defective inter-domain interaction.…”