title: Skeletal muscle ryanodine receptor mutations associated with malignant hyperthermia showed enhanced intensity and sensitivity to triggering drugs when expressed in human embryonic kidney cells.
abstract: BACKGROUND
  Mutations within the gene encoding the skeletal muscle calcium channel ryanodine receptor can result in malignant hyperthermia. Although it is important to characterize the functional effects of candidate mutations to establish a genetic test for diagnosis, ex vivo methods are limited because of the low incidence of the disorder and sample unavailability. More than 250 candidate mutations have been identified, but only a few mutations have been functionally characterized.
  
  
  METHODS
  The human skeletal muscle ryanodine receptor complementary DNA was cloned with or without a disease-related variant. Wild-type and mutant calcium channel proteins were transiently expressed in human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40, and functional analysis was carried out using calcium imaging with fura-2 AM. Six human malignant hyperthermia-related mutants such as R44C, R163C, R401C, R533C, R533H, and H4833Y were analyzed. Cells were stimulated with a specific ryanodine receptor agonist 4-chloro-m-cresol, and intracellular calcium mobility was analyzed to determine the functional aspects of mutant channels.
  
  
  RESULTS
  Mutant proteins that contained a variant linked to malignant hyperthermia showed higher sensitivity to the agonist. Compared with the wild type (EC50=453.2 µM, n=18), all six mutants showed a lower EC50 (21.2-170.4 µM, n=12-23), indicating susceptibility against triggering agents.
  
  
  CONCLUSIONS
  These six mutations cause functional abnormality of the calcium channel, leading to higher sensitivity to a specific agonist, and therefore could be considered potentially causative of malignant hyperthermia reactions.
authors: Department of Medicine, University of Florida, Gainesville, Florida, USA.
authors: Keisaku
authors: K
authors: Sato
authors: Cornelia
authors: C
authors: Roesl
authors: Neil
authors: N
authors: Pollock
authors: Kathryn M
authors: KM
authors: Stowell
mesh_terms: D002118:Calcium / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D008305:Malignant Hyperthermia / Q000235:genetics* / Q000503:physiopathology; D009154:Mutation / Q000235:genetics* / Q000502:physiology; D059906:Neuroimaging; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology
chemical_list: D018076:DNA, Complementary; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium