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Luminal accessory protein regulation of wild type and mutant human cardiac ryanodine receptors (hRyR2)

Maxwell, Chloe 2014. Luminal accessory protein regulation of wild type and mutant human cardiac ryanodine receptors (hRyR2). PhD Thesis, Cardiff University.
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Human cardiac ryanodine receptors (hRyR2) are major Ca2+ release channels in the heart, which form quaternary complexes with luminal proteins, calsequestrin (CSQ2), junctin (JUN) and triadin (TRD1). These proteins facilitate Ca2+ release during excitation-contraction coupling, modulating the response of hRyR2 to luminal Ca2+ changes. Catecholaminergic Polymorphic Ventricular Tachycardia is an arrhythmogenic disorder, caused by mutations in RyR2 and CSQ2 genes. Defective sensing of cytosolic/luminal Ca2+ by hRyR2 is a candidate mechanism underlying disease pathogenesis, likely caused by defective luminal protein regulation. Using a recombinant approach, this study aimed to evaluate if mutant (N4104K and A4556T) hRyR2 respond to or interact differently with, these accessory proteins. Expression constructs corresponding to CSQ2 and JUN were generated and expressed stably and transiently with wild-type (WT) or mutant hRyR2 in HEK293 cells. Immunofluorescent co-localisation confirmed successful co-expression and association of these luminal proteins with hRyR2 in situ. Ca2+ activation of wild-type/mutant hRyR2 in the absence/presence of CSQ2 and/or JUN was assessed by [3H]-ryanodine binding, while luminal Ca2+ effects were monitored using single-cell Ca2+ imaging - examining spontaneous Ca2+ release events. A4556T-hRyR2 displayed similar cytosolic and luminal Ca2+ dependence to wild-type channels, whilst N4104K-hRyR2 displayed a remarkably different Ca2+ activation profile, demonstrating functional heterogeneity between hRyR2 mutants. Ca2+ imaging revealed an inhibitory effect of CSQ2 on WT and N4104K-hRyR2 activity, both in the presence and absence of JUN. In line with this, CSQ2 was found to bind directly to hRyR2 by co-immunoprecipitation, an observation that has not been previously demonstrated in the literature. Immunofluorescence studies suggested reduced CSQ2 and JUN association with A4556T-hRyR2, but this could not be confirmed with co-immunoprecipitation. Ca2+ imaging investigations with this mutant however, suggested that CSQ2 wasn’t able to regulate these channels in the same way as WT, implying a change in functional effect.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 05 Jun 2017 04:53

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