A functional analysis of RYR1 mutations causing malignant hyperthermia : a thesis presented to Massey University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry
Malignant hyperthermia (MH) is a rare pharmacogenetic disorder in humans induced by
volatile anaesthetics and depolarising muscle relaxants. An MH reaction shows
abnormal calcium homeostasis in skeletal muscle leading to a hypermetabolic state and
increased muscle contracture. A mutation within the skeletal muscle calcium release
channel ryanodine receptor gene (RYR1) is associated with MH and is thought to cause
functional defects in the RYR1 channel leading to abnormal calcium release to the
sarcoplasm and consequent MH reactions. Mutations within RYR1 are also associated
with a rare congenital myopathy, central core disease (CCD). CCD is characterised by
muscle weakness and is thought to be caused by insufficient calcium release from the
RYR1 channel during excitation-contraction (EC) coupling.
To investigate functional effects of RYR1 mutations, the entire coding region of human
RYR1 was assembled and cloned into an expression vector. Mutant clones containing
RYR1 mutations linked to MH or CCD were also constructed. Wild-type (WT) and
mutant RYR1 clones were used for transient transfection of HEK-293 cells. Western
blotting was performed after harvesting and expressed WT and mutant RYR1 proteins
were successfully detected. Immunofluorescence showed co-localisation of RYR1
proteins and the endoplasmic reticulum in HEK-293 cells. [3H]ryanodine binding assays
showed that RYR1 mutants linked to MH were more sensitive to the agonist
4-chloro-m-cresol (4-CmC) and less sensitive to the antagonist Mg2+ compared with WT.
Two C-terminal RYR1 mutants T4826I and H4833Y were very significantly
hypersensitive to 4-CmC and they may also result in a leaky channel. This
hypersensitivity of mutants linked to MH may result in abnormal calcium release
through the RYR1 channel induced by triggering agents leading to MH reactions. RYR1
mutants linked to CCD showed no response to 4-CmC showing their hyposensitive
characteristics to agonists.
This study showed that the human RYR1 proteins could be expressed in HEK-293 cells.
Moreover, using the recombinant human RYR1 clone, a single mutation within RYR1
resulted in a functional defect in expressed RYR1 proteins and functions of mutant
RYR1 proteins varied from hypersensitive to hyposensitive depending on the mutation
and whether it was linked to MH or CCD.