Defects in the Ca²§+ release channel of skeletal muscle sarcoplasmic reticulum that are associated with malignant hyperthermia and central core disease

grantor: University of TorontoMalignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, seventeen mutations in the human RYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor or RyR1) have been associated with MH and/or CCD. The hypotheses tested in this thesis was that MH and CCD mutations increase the sensitivity of mutant Ca2+ release channels to caffeine and halothane; that MH and CCD mutations increase the permeability (leakiness) of the mutant channels; and that CCD mutant channels are more leaky than MH channels. Sixteen mutations corresponding to human MH or CCD mutations were made in three distinct regions in a full length rabbit RYR1 cDNA. Ca 2+ release, monitored by Fura-2, in HEK-293 cells expressing MH or CCD mutant ryanodine receptors was more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild-type receptors. Linear regression analysis showed a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical ' in vitro' caffeine halothane contracture test (IVCT). Resting Ca 2+ concentrations were higher and maximal peak amplitudes of caffeine-induced Ca2+ release were lower in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1, supporting the hypothesis that CCD mutant channels are more leaky. A CCD mutation in the transmembrane/lumenal domain of RyR1, 14897T, was so permeable that its altered function could only be demonstrated in heterotetrameric form. The content of endogenous sarco(endo)plasmic reticulum Ca2+ ATPase isoform 2b (SERCA2b), measured by ELISA, 45Ca 2+ uptake and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that ER Ca 2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. These results support the hypothesis that MH/CCD mutant channels are more sensitive to triggering anesthetics than wild type channel, and that CCD mutant channels have higher Ca2+ permeability than MH mutant channels.Ph.D