DANTROLENE

Dantrolene is the drug of choice for preventing and reversing the symptoms of MH. Dantrolene sodium is a hydantoin derivative (1-[[[5-(4-nitrophenyl)-2-furanyl]methylene]imino]-2,4-imidazolidinedione) that relaxes but does not totally paralyze skeletal muscle. These properties of dantrolene have been closely correlated with its ability to reduce Ca²⁺ efflux from SR in vitro. Dantrolene (20 µM) counteracts the effect of reduced Mg²⁺ inhibition in MH-affected muscle.^[38] Caffeine contractures induced after K⁺ conditioning of porcine skeletal muscle were found to render muscles refractory to brief electrical stimulation, but there was still an enhancement of contracture tension elicited by subsequent direct caffeine stimulation of SR calcium release. This enhanced sensitivity to caffeine was inhibited by dantrolene (20 µM) and its water-soluble analog azumolene (150 µM).

Preparations of skeletal SR membrane vesicles have been used to examine the ability of dantrolene to alter Ca²⁺ fluxes. Dantrolene (10 to 90 µM) was shown to inhibit SR Ca²⁺ release, especially when assayed in the presence of caffeine and adenine nucleotide. Later, depolarization-induced

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Experiments of radioligand-receptor binding performed with [³ H]ryanodine have shown that under certain assay conditions, micromolar concentrations of dantrolene or its water-soluble derivative azumolene could inhibit the binding of ryanodine to its conformationally sensitive site. In this respect, doxorubicin-stimulated binding was much more inhibited by dantrolene than by caffeine or Ca²⁺ -stimulated binding. These results are in agreement with Ca²⁺ transport studies with skeletal SR, in which azumolene was shown to block doxorubicin-induced Ca²⁺ release. However, a later investigation found little pharmacologic overlap between the modulation of [³ H]ryanodine and [³ H]dantrolene binding sites in porcine skeletal muscle. For example, the binding of [³ H]dantrolene was insensitive to ryanodine and Ca²⁺ and adenine nucleotides. Experiments performed with [³ H]dantrolene have revealed that specific binding sites for the drug colocalize to junctional SR membranes with [³ H]ryanodine binding sites. [³ H]Dantrolene binding sites were not detected in T-tubule membranes or sarcolemmal membranes.

The idea that dantrolene suppresses SR Ca²⁺ release as a result of direct interactions with RYR1 has been somewhat controversial. Significant progress has been made to positively identify the location of dantrolene binding sites within the EC coupling machinery. Paul-Pletzer and associates^[41] demonstrated that [³ H]azidodantrolene, a pharmacologically active, photoaffinity analog of dantrolene, specifically labels the amino terminus of RYR1. The [³ H]azidodantrolene binding site was localized to the 1400-amino acid residue fragment of RYR1 cleaved by n-calpain, a tissue-specific isoform of this Ca²⁺ and thiol-activated protease. More detailed analysis further localized the [³ H]azidodantrolene binding site to a single domain containing the core sequence corresponding to amino acid residues 590 through 609 of RYR1.^[42] Evidence of the specificity of the [3H]azidodantrolene photoaffinity-labeling procedure was provided based on the observation that a monoclonal antibody that recognizes the 172-kd, n-calpain-cleaved, amino-terminal fragment inhibited [³ H]azidodantrolene photolabeling of RYR1 in SR in a concentration-dependent manner. Ikemoto and coworkers^{[43] [44]} proposed that this very region of the RYR1 structure (DP1 domain) participates in interdomain interactions that stabilize the closure of the Ca²⁺ channel state. Based on this model, one possible mechanism by which dantrolene inhibits release of SR Ca²⁺ is by directly binding to the DP1 region and stabilizing the interdomain interaction of DP1.^[42] How dantrolene binding to the DP1 domain stabilizes rather than destabilizes interdomain interactions remains a mystery.