Dantrolene rescues aberrant N-terminus intersubunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors

Seidel, Monika, Thomas, N. Lowri ORCID: https://orcid.org/0000-0001-8822-8576, Williams, Alan J., Lai, F. Anthony and Zissimopoulos, Spyros 2015. Dantrolene rescues aberrant N-terminus intersubunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors. Cardiovascular Research 105 (1) , pp. 118-128. 10.1093/cvr/cvu240

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Abstract

Aims The ryanodine receptor (RyR2) is an intracellular Ca2+ release channel essential for cardiac excitation–contraction coupling. Abnormal RyR2 channel function results in the generation of arrhythmias and sudden cardiac death. The present study was undertaken to investigate the mechanistic basis of RyR2 dysfunction in inherited arrhythmogenic cardiac disease. Methods and results We present several lines of complementary evidence, indicating that the arrhythmia-associated L433P mutation disrupts RyR2 N-terminus self-association. A combination of yeast two-hybrid, co-immunoprecipitation, and chemical cross-linking assays collectively demonstrate that a RyR2 N-terminal fragment carrying the L433P mutation displays substantially reduced self-interaction compared with wild type. Moreover, sucrose density gradient centrifugation reveals that the L433P mutation impairs tetramerization of the full-length channel. [3H]Ryanodine-binding assays demonstrate that disrupted N-terminal intersubunit interactions within RyR2L433P confer an altered sensitivity to Ca2+ activation. Calcium imaging of RyR2L433P-expressing cells reveals substantially prolonged Ca2+ transients and reduced Ca2+ store content indicating defective channel closure. Importantly, dantrolene treatment reverses the L433P mutation-induced impairment and restores channel function. Conclusion The N-terminus domain constitutes an important structural determinant for the functional oligomerization of RyR2. Our findings are consistent with defective N-terminus self-association as a molecular mechanism underlying RyR2 channel deregulation in inherited arrhythmogenic cardiac disease. Significantly, the therapeutic action of dantrolene may occur via the restoration of normal RyR2 N-terminal intersubunit interactions.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Pharmacy Medicine Biosciences
Subjects:	R Medicine > R Medicine (General)
Publisher:	Oxford University Press (OUP): Policy B
ISSN:	0008-6363
Funders:	British Heart Foundation
Date of First Compliant Deposit:	8 March 2019
Date of Acceptance:	27 October 2014
Last Modified:	08 May 2023 07:41
URI:	https://orca.cardiff.ac.uk/id/eprint/69651

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