Characterisation RyR1 variants linked to malignant hyperthermia : a thesis presented to Massey University in partial fulfilment of the requirements for a Masters of Science in Biochemistry

Abstract

Malignant hyperthermia is a potentially fatal disorder of skeletal muscle manifesting as a rise in body temperature in response inhalational anaesthetics and muscle relaxants. Further clinical signs include muscle rigidity and increased oxygen consumption. The increased metabolism is induced by alterations to Ca2+ homeostasis resulting from the dysregulation of the sarcoplasmic reticulum protein the ryanodine receptor type 1 (RyR1). A large proportion of known malignant hyperthermia linked genetic variants reside within the gene encoding the type 1 ryanodine receptor, RYR1. Malignant hyperthermia can be diagnosed by in vitro contracture testing of biopsied muscle tissue. The use of DNA diagnostic testing is advantageous, however it is limited to only 35 of the proposed 400 RYR1 linked variants known to be associated with malignant hyperthermia. The research described in this thesis reports the functional characterisation of two RYR1 variants linked to malignant hyperthermia, c.641C>T and c.7042_7044delCAG resulting in the amino acid changes p.T214M and p.ΔE2348. The ability of each variant to release Ca2+ in response to a stimulus was examined in a heterologous system. The variant p.ΔE2348 was shown to be hyperactive in response to agonists indicating the variant is the cause of malignant hyperthermia, while the p.T214M variant does not appear to have an effect ryanodine receptor function. To understand the relationship between RyR1 function and any structural alterations induced by the p.T214M and p.ΔE2348 variants, the domain housing each variant was cloned for bacterial expression. Subsequent purification and structural characterisation could be used to explain the role each variant plays with respect to the onset of MH. The RyR1 N-­terminal domain, amino acids 1-­558, and helical domain, amino acids 2091-­2525, were expressed in E. coli and partially purified. The domains were shown to be soluble and stable following expression.