Functional defects in six ryanodine receptor isoform-1 (RyR1) mutations associated with malignant hyperthermia and their impact on skeletal excitation-contraction coupling

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 (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) 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 Ca2+ 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 MHRyR1s are therefore related to the nature of the stimulus rather than size of the Ca2+ store. The functional changes of (MH)RyR1s were directly analyzed using [H-3] ryanodine binding analysis of isolated myotube membranes. Although none of the MHRyR1s examined significantly altered EC50 for Ca2+ activation, many failed to be completely inhibited by a low Ca2+ (less than or equal to100 nM), and all were significantly more responsive to caffeine than (WT)RyR1 at Ca2+ concentrations that approximate those in resting myotubes. All seven mutations had diminished sensitivity to inhibition by Ca2+ and Mg2+. 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 Ca2+ and Mg2+ inhibition and inability of MHRyR1s to be fully inactivated at [Ca2+](i) typical of normal myotubes at rest are key defects that contribute to the initiation of MH episodes.