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ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor

Blayney, Lynda Mary, Beck, Konrad, MacDonald, Ewan, D'Cruz, Leon G,, Nomikos, Michail, Griffiths, Julia, Thanassoulas, Angelos, Nounesis, George and Lai, Francis Anthony 2013. ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor. Biochimica et Biophysica Acta (BBA) - General Subjects 1830 (10) , pp. 4426-4432. 10.1016/j.bbagen.2013.05.038

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Abstract

BACKGROUND: This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). METHODS: Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. RESULTS: The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. CONCLUSIONS: The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. GENERAL SIGNIFICANCE: Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Dentistry
Medicine
Subjects: Q Science > QP Physiology
R Medicine > R Medicine (General)
R Medicine > RM Therapeutics. Pharmacology
Uncontrolled Keywords: Cardiac ryanodine receptor; ATP binding site; ATP binding motifs; FKBP12.6; Catecholaminergic polymorphic ventricular tachycardia (CPVT) mutations; Caffeine
Publisher: Elsevier
ISSN: 0304-4165
Funders: MRC studentship to Julia Griffith, BHF project grant (PG/05077) to Francis Anthony Lai
Last Modified: 27 Mar 2020 02:38
URI: http://orca.cf.ac.uk/id/eprint/51494

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