title: 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.
abstract: 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 < 0.001) 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+ photometry assay in the assessment of the sensitivity to caffeine and halothane of specific ryanodine receptor mutants.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Charles H. Best Institute, Toronto, Ontario M5G 1L6, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Tong
authors.forename: H
authors.initials: H
authors.lastname: Oyamada
authors.forename: N
authors.initials: N
authors.lastname: Demaurex
authors.forename: S
authors.initials: S
authors.lastname: Grinstein
authors.forename: T V
authors.initials: TV
authors.lastname: McCarthy
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary; D006221:Halothane / Q000494:pharmacology*; D006801:Humans; D008305:Malignant Hyperthermia / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D017696:Myopathies, Nemaline / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium; D006221:Halothane