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Title: Molecular mechanisms underlying cardiac ryanodine receptor dysfunction in sudden cardiac death
Author: Thomas, Nia Lowri
ISNI:       0000 0004 2746 389X
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2005
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Ca2+ release via the cardiac ryanodine receptor (RyR2) is a fundamental event in excitation-contraction coupling. Point mutations in the gene encoding RyR2 are associated with arrhythmogenic right ventricular dysplasia type 2 (ARVD2), a disease likely characterised by abnormal release of Ca2+ that may result in sudden death. GFP-tagged RyR2 mutants (R176Q/T2504M, L433P and N2386I) were generated and expressed in a human embryonic kidney (HEK) cell model, enabling profiling of the amplitude and temporal characteristics of caffeine-evoked Ca2+ release through homotetrameric channels using confocal microscopy. Mutants were functionally heterogeneous and demonstrated profound differences in Ca2+ release when compared with WT channels, including the novel observation that one of the mutants (L433P) exhibited reduced sensitivity to caffeine activation. The molecular basis of this heterogeneity was investigated by determining the sensitivity of the mutant channels to cytoplasmic Ca2+. This was achieved by evaluation of caffeine-induced Ca2+ release from WT or mutants channels in streptolysin-O permeabilised HEK cells, where the cytoplasmic Ca2+ concentration was clamped. Although resting ER Ca2+ store and cytoplasmic Ca2+ levels were comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca2+-dependent inactivation. We also investigated whether these mutations disrupted the interaction between RyR2 and accessory proteins involved in normal channel function. cDNA encoding mutation susceptible regions were constructed and screened against a human cardiac cDNA library using a yeast two hybrid system. The N2386I mutation abolished association of the RyR2 domain with two cardiac proteins, which robustly occurred with the corresponding WT domain. These findings demonstrate that ARVD2-linked RyR2 mutations critically affect channel activation and suggest that differential sensitivity to cytoplasmic Ca2+ may be a causative mechanism in the pathogenesis of this disease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: R Medicine (General)