Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MH RyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WT RyR1, six of the most common human MH RyR1s (Arg163Cys,

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... Arg, Arg614Cys, Arg2163Cys, Val2168Met, and Arg2458His), and a newly identified C-terminal mutation (Thr4826Ile) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MH RyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WT RyR1. The hypersensitivity of MH myotubes extended to K + depolarization. MH myotubes responded to direct channel activators with maximum Ca 2+ amplitudes consistently smaller than WT myotubes, whereas the amplitude of their responses to depolarization were consistently larger than WT myotubes. The magnitudes of responses attainable from myotubes expressing MH RyR1s are therefore related to the nature of the stimulus rather than size of the Ca 2+ store. The functional changes of MH RyR1s were directly analyzed using [ 3 H]ryanodine binding analysis of isolated myotube membranes. Although none of the MH RyR1s examined significantly altered EC 50 for Ca 2+ activation, many failed to be completely inhibited by a low Ca 2+ (<100 nM), and all were significantly more responsive to caffeine than WT RyR1 at Ca 2+ concentrations that approximate those in resting myotubes. All seven mutations had diminished sensitivity to inhibition by Ca 2+ and Mg 2+ . Using a homologous expression system, our study demonstrates for the first time that these 7 MH mutations are all both necessary and sufficient to induce MH related phenotypes. Decreased sensitivity to Ca 2+ and