title: Functional characterisation of the R2452W ryanodine receptor variant associated with malignant hyperthermia susceptibility.
abstract: Malignant hyperthermia (MH) is a pharmacogenetic disorder that manifests in susceptible individuals exposed to volatile anaesthetics. Over 400 variants in the ryanodine receptor 1 (RYR1) have been reported but relatively few have been definitively associated with susceptibility to MH. This is largely due to the technical challenges of demonstrating abnormal Ca(2+) release from the sarcoplasmic reticulum. This study focuses on the R2452W variant and its functional characterisation with the aim of classifying this variant as MH causative. HEK293 cells were transiently transfected with full-length human wildtype or R2452W mutant RYR1 cDNA. In addition, B-lymphoblastoid cells from blood and myoblasts propagated from in vitro contracture tests were extracted from patients positive for the R2452W variant. All cell lines generated were loaded with the ratiometric dye Fura-2 AM, stimulated with the RYR1-specific agonist 4-chloro-m-cresol and Ca(2+) release from the sarcoplasmic/endoplasmic reticulum was monitored by fluorescence emission. All cells expressing the RYR1 R2452W variant show a significantly higher Ca(2+) release in response to the agonist, 4-chloro-m-cresol, compared to cells expressing RYR1 WT. These results indicate that the R2452W variant results in a hypersensitive ryanodine receptor 1 and suggest that the R2452W variant in the ryanodine receptor 1 is likely to be causative of MH.
authors.affiliation: Centre for Reproductive Health, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Roesl
authors.affiliation: Department of Medicine, University of Florida, Gainesville, FL, United States.
authors.forename: Keisaku
authors.initials: K
authors.lastname: Sato
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: Anja
authors.initials: A
authors.lastname: Schiemann
authors.affiliation: Department of Anaesthesia and Intensive Care, Palmerston North Hospital, Palmerstion North, New Zealand.
authors.forename: Neil
authors.initials: N
authors.lastname: Pollock
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. Electronic address: k.m.stowell@massey.ac.nz.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Stowell
mesh_terms: D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D003408:Cresols / Q000494:pharmacology; D004198:Disease Susceptibility; D005260:Female; D005455:Fluorescent Antibody Technique; D016257:Fura-2; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008305:Malignant Hyperthermia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009154:Mutation / Q000235:genetics*; D010375:Pedigree; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism
chemical_list: D000697:Central Nervous System Stimulants; D003408:Cresols; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; C032538:4-cresol; D002110:Caffeine; D002118:Calcium; D016257:Fura-2
keywords: Calcium; Malignant hyperthermia; Ryanodine receptor; Skeletal muscle