Ryanodine receptors (RyRs) are the largest known ion channels composed of four identical subunits. Interactions between structural/functional domains have been proposed to regulate channel activity and play an important role in the pathogenesis of RyR-associated disorders. RyR2 mediates the release of calcium form sarcoplasmic reticulum of cardiac myocytes and its dysfunction is associated with life-threatening arrhythmias. The principal aim of this study was to characterise the self-association of the RyR2 N-terminus biochemically and evaluate its impact on channel function. Moreover, its role in channel dysfunction observed in arrhythmia-susceptible individuals was tested together with dantrolene’s ability to rescue the disease phenotype. RyR2 N-terminus self-association is mediated by multiple sites with two critical oligomerisation determinants located in the loops connecting strands β8-β9 and β20-β21, predicted to reside at the inter-subunit interface. N-terminus self-association is further stabilised by disulphide bonds most likely involving multiple cysteine residues with cysteine 361 contributing to this process. Normal N-terminal inter-subunit interactions within the full-length RyR2 appear to prevent spontaneous activation of the channel at diastolic calcium. Channel hypersensitivity is a common feature of the arrhythmia-associated phenotype suggesting that abnormal N-terminus self-interaction might be involved in RyR2 pathology. Indeed the presence of arrhythmia-linked mutations (L433P and R176Q) compromises the ability of the RyR2 N-terminus to oligomerise. Defective N-terminus self-association appears to underlie the functional impairment of RyR2L433P. The mutated channel displays compromised [3H]ryanodine binding and reduced stability of tetrameric assembly, both of which can be rescued by dantrolene at clinically relevant concentrations. Notably, dantrolene’s primary mode of action appears to involve stabilisation of the N-terminal inter-subunit interactions. In summary, the work presented here provides important insights into a novel domain-domain interaction and its role in the regulation of RyR2 function.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:590381 |
| Date | January 2014 |
| Creators | Seidel, Monika |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/57357/ |
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