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 _MHRyR1s 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 _WTRyR1, six of the most common human _MHRyR1s (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 _MHRyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing _WTRyR1. 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 _MHRyR1s were directly analyzed using [³H]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⁺ (≤100 nm), and all were significantly more responsive to caffeine than _WTRyR1 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.