The cellular Ca2+ photometry assay demonstrates abnormal sensitivity to caffeine and halothane in 15 RYR1 mutations, supporting their causal role in malignant hyperthermia.
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 lod (log of the odds favoring linkage versus nonlinkage) scores, 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 transfected 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 0.05). 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+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants.
Tong et al. (1997) studied this question.