Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease

Malignant 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, 17 mutations in the human RYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor) have been associated with MH and/or CCD, Although many of these mutations have been linked to MH and/or CCD, with high Iod (log of the odds favoring linkage versus nonlinkage) scopes, others have been found in single, small families, Independent biochemical evidence for a causal role for these mutations in MH is available for only two mutants, Mutations corresponding to the human MH mutations were made in a full-length rabbit RYR1 cDNA, and wild type. and mutant cDNAs were transected into HEK-293 cells, After about 48 h, intact cells were loaded with the fluorescent Ca2+ indicator, fura-2, and intracellular Ca2+ release, induced by caffeine or halothane, was measured by photometry. Ca2+ release in cells expressing MH or CCD mutant ryanodine receptors was invariably significantly more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild type receptors or receptors mutated in other regions of the molecule, Linear regression analysis showed that there is 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). The correlation was weaker, however for halothane (r = 0.49, p > 0.05), Abnormal sensitivity in the Ca2+ photometry assay provides supporting evidence for a causal role in MH for each of 15 single amino acid mutations in the ryanodine receptor, The study demonstrates the usefulness of the cellular Ca2+ photometpy assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants.