Functional and structural characterisation of the malignant hyperthermia associated RYR1 mutation R245W : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry

Abstract

Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle triggered by volatile halogenated anaesthetics. Susceptible individuals can exhibit symptoms including tachycardia, high temperature, hypoxia, hypermetabolism and skeletal muscle rigidity. There are usually no symptoms of MH in normal day to day life although “awake” episodes have been reported as a result of extreme exercise in MH-susceptible subjects. Genetic variants have been associated with the skeletal muscle ryanodine receptor gene (RYR1) in 50-70 % of susceptible patients. At the molecular level susceptible patients are thought to have an increased sensitivity to RYR1 agonists in skeletal muscle compared to non-affected patients that results in disregulated skeletal muscle Ca2+ homeostasis. In 2000 a novel RYR1-mutation, c.G7354T (p.R2452W), associated with MH, was identified in a New Zealand (NZ) family. Subsequently the same mutation was identified in a separate NZ family and has also been reported in the United Kingdom. To date this mutation had not been shown to be causative of MH. Therefore, the aim of this study was to carry out functional analyses to test whether the R2452W mutant receptor alters Ca2+ release compared to wildtype. For this study Ca2+ release assays were carried out in three different cell types: B-lymphoblastoid cells, myotubes and HEK293 cells transfected with full-length human RYR1 cDNA. Cells were exposed to the RYR1-specific agonist 4-chloro-m-cresol, which stimulates Ca2+ release through the receptor while the increase in cytosolic Ca2+ was detected using a membrane-permeable fluorophore. Cells expressing R2452W mutant RYR1 showed altered Ca2+ release from the sarco(endo)plasmic reticulum suggesting a hypersensitive channel. In order to study structure/function relationships of the R2452W mutation within the RYR1 protein, as well as the 3D structure of a central RYR1 domain (amino acid 2144-2489), a region encompassing this substitution was cloned for bacterial expression and subsequently purified. Wildtype and mutant proteins were compared to determine any effects the mutation may have upon the stability of the protein. Wildtype and R2452W RYR1 protein showed no obvious differences in stability but both proteins appeared to oligomerise suggesting this region might be involved in RYR1-RYR1 domain interactions.