The L433P Arrhythmia-Linked Mutation Disrupts Amino-Terminus Oligomerisation of the Human Cardiac Ryanodine Receptor

Abstract

The type 2 ryanodine receptor (RyR2) mediates calcium release from the sarcoplasmic reticulum of cardiomyocytes. RyR2 mutations found in three clusters including the amino-terminus are associated with arrhythmogenic cardiac disease. Arrhythmia-linked mutations are proposed to disrupt interactions between discrete functional domains within the RyR2 tetramer, resulting in abnormal channel gating. Recently, we presented evidence that the RyR2 N-terminus self-associates into a tetrameric form, which stabilises the closed channel conformation. Here, we report that the arrhythmia-associated L433P mutation affects oligomerisation of the RyR2 N-terminus. Tetramerisation ability was tested by chemical cross-linking experiments of an RyR2 N-terminal fragment (residues 1-906) containing L433P, expressed in HEK293 cells. The mutant fragment displayed reduced ability for tetramerisation versus wild-type. Two additional techniques, the yeast two-hybrid system and co-immunoprecipitation assays, further indicated that the mutant fragment displays reduced self-interaction and reduced binding to wild-type N-terminus. Notably, dantrolene, a drug used to treat the clinical symptoms of malignant hyperthermia and whose target-binding site lies within the RyR N-terminus, was able to partially restore the tetramer in the L433P mutant. The effect of the L433P mutation was also investigated in the context of the full-length channel. We performed sucrose density gradient centrifugation of CHAPS-solubilised RyR2 to assess the relative stability of tetrameric functional channels. We found that wild-type RyR2 remains a tetramer consistent with an intact functional channel, whereas a substantial proportion of RyR2/L433P channels is dissociated into monomers. Our findings suggest that disruption of inter-subunit interactions within the N-terminus of mutant RyR2 might contribute to the mechanism by which some of the arrhythmia-associated mutations result in RyR2 channel dysfunction.