Molecular transitions regulating ryanodine receptor channel gating: Potential target for therapeutic intervention in heart failure and arrhythmia

Griffiths, Julia 2011. Molecular transitions regulating ryanodine receptor channel gating: Potential target for therapeutic intervention in heart failure and arrhythmia. PhD Thesis, Cardiff University.

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Abstract

Aberrant calcium handling in cardiomyocytes, linked to ryanodine receptor (RyR) dysfunction, causes cardiac arrhythmias underlying catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure. CPVT links to point mutations in specific domains of the cardiac ryanodine receptor (RyR2). Similar mutational clusters in RyR1 (skeletal muscle) cause malignant hyperthermia and central core disease suggesting common mechanisms of dysfunction underlying cardiac and skeletal muscle pathologies, leading to the domain interaction hypothesis. Peptides complimentary to N-terminus and central domain mutational clusters activate RyR by disrupting interaction. RyR1/2 is regulated by calcium, ATP, magnesium, phosphorylation and the accessory protein FKBP12/12.6. Previous work, on RyR1/2 modulation, assessing phosphorylation in relation to FKBP12/12.6 stabilisation of channel activity, indicates convergent regulation involving control of domain interaction sites by phosphorylation, FKBP12/12.6 and other modulators. This study used a central domain peptide (DP4) and its mutant form (DP4M) to investigate the relationship between FKBP12, phosphorylation, and other modulators to identify this region of convergent regulation. Native RyR1 channels, stripped of endogenous FKBP12 and phosphorylated or dephosphorylated, were shown, by [3H]ryanodine binding, to be fully functional and to be activated by calcium and ATP. FKBP12 and magnesium were each shown to inhibit channel activity, and phosphorylation reversed magnesium inhibition. DP4, peptide, synthesised from two sources, activated RyR1 2-fold in the presence of pM calcium, less than the activity observed with ATP. Magnesium and FKBP12 inhibited RyR1 activation by DP4. In contrast to previous studies, DP4M was not an inactive control peptide. To investigate this discrepancy, recombinant GST-tagged (R-)DP4M and (R-)DP4 were generated and neither showed an activatory effect. However, once cleaved of GST, the R-DP4 was as potent as synthetic DP4 at 10 pM. Experiments with RyR2 demonstrated similar results for calcium and ATP activation with inhibition by FKBP12.6. The RyR2 central domain peptide (DPc10) has been cloned and the protein produced.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
Subjects:	R Medicine > RC Internal medicine
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Mar 2016 23:29
URI:	https://orca.cardiff.ac.uk/id/eprint/54156

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