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Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function

Seidel, Monika, de Meritens, Camille Rabesahala, Johnson, Louisa, Parthimos, Dimitris, Bannister, Mark, Thomas, N. Lowri, Ozekhome-Mike, Esizaze, Lai, F. Anthony and Zissimopoulos, Spyros 2020. Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function. Cardiovascular Research , cvaa043. 10.1093/cvr/cvaa043

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Abstract

Aims The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer. Methods and Results We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the β8 with β9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively-charged side chains of two aspartate residues (D179 and D180) within the β8-β9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The β8-β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8-β9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the β8-β9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the β8-β9 domain in channel closing. Conclusions These results suggest that efficient N-terminus inter-subunit communication mediated by the β8-β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression. Translational Potential Our findings that the RyR2 β8-β9 loop is involved in both Ca2+ release channel opening and closing have important clinical implications. This RyR2 domain is a known “hot-spot” for mutations associated with arrhythmogenic cardiac disease, which could produce hypersensitive as well as hyposensitive channels. Therapeutic strategies currently focus on gain-of-function RyR2 channels to suppress sarcoplasmic reticulum Ca2+ release either indirectly with class I/II anti-arrhythmic drugs, or by directly targeting RyR2 to inhibit channel activity. These strategies may not only be ineffective, but they may exacerbate the malignant phenotype in the case of loss-of-function RyR2 mutations.

Item Type: Article
Date Type: Published Online
Status: In Press
Schools: Pharmacy
Medicine
Biosciences
Publisher: Oxford University Press (OUP): Policy B
ISSN: 0008-6363
Date of First Compliant Deposit: 10 March 2020
Date of Acceptance: 12 February 2020
Last Modified: 31 Aug 2020 15:31
URI: http://orca-mwe.cf.ac.uk/id/eprint/130288

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