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Functional heterogeneity of CPVT-mutant human cardiac ryanodine receptors: evaluation of the influence of S2031 and S2808 phosphorylation sites

Hamilton, Shanna 2017. Functional heterogeneity of CPVT-mutant human cardiac ryanodine receptors: evaluation of the influence of S2031 and S2808 phosphorylation sites. PhD Thesis, Cardiff University.
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Human cardiac ryanodine receptors (hRyR2) are calcium (Ca2+) release channels central to excitation-contraction coupling. Mutations in hRyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), a genetic disorder characterized by arrhythmia, occurring under adrenergic drive. Recent studies suggest gain-of-function mutant channels may undergo different mechanisms of dysfunction, with some showing altered Ca2+ release under basal conditions and others requiring an additional trigger (possibly in the form of β-adrenergic phosphorylation). This study evaluated whether mutants from different domains of hRyR2 (S2246L and N4104K) were functionally heterogeneous in a cellular setting and whether this was related to phosphorylation status. Cells (human embryonic kidney 293) expressing N4104K-hRyR2 displayed smaller, faster Ca2+ release events than those expressing the wild type (WT), while those of cells expressing S2246L-hRyR2 were similar to WT. However, a lower proportion of S2246L cells showed any kind of Ca2+ release functionality compared to those expressing WT or N4104K, reinforcing that these mutations cause different types of dysfunction. Assessment of phosphorylation status using site-specific antibodies for protein kinase A (PKA) target sites S2808 and S2031 showed that mutant phosphorylation levels were different to each other and to that of the WT, indicating a possible role of phosphorylation in this. Activation of PKA-mediated phosphorylation in cells using a cyclic AMP analogue resulted in changes to the kinetics of spontaneous Ca2+ release via WT hRyR2, but did not significantly affect that of mutants. Genetic phosphorylation at either PKA site (S2808D and S2031D) altered the Ca2+ release of mutants as well as WT hRyR2, but this was found to be due to changes in expression of sarco-endoplasmic reticulum Ca2+-ATPase (SERCa) pump, which also contributes to Ca2+ homeostasis. These findings reinforce the concept that phosphorylation is linked to dysfunction and contributes to our understanding of hRyR2 mutation in arrhythmia, highlighting that ‘gain-of-function’ does not necessarily equate to dysfunction via the same mechanism.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Funders: British Heart Foundation
Date of First Compliant Deposit: 14 May 2018
Last Modified: 14 May 2018 09:08

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