Monika Seidel1,2, Camille Rabesahala de Meritens1,2, Louisa Johnson1,2, Dimitris Parthimos1,3, Mark Bannister1,2, Nia Lowri Thomas1,4, Esizaze Ozekhome-Mike1,2, Francis Anthony Lai5, Spyros Zissimopoulos1,2. 1. Department of Cardiology, School of Medicine, Wales Heart Research Institute, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. 2. Swansea University Medical School, Institute of Life Science, Swansea SA2 8PP, UK. 3. Division of Cancer & Genetics, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK. 4. School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Cardiff CF10 3NB, UK. 5. College of Medicine, QU Health, and Biomedical Research Centre, Qatar University, Doha, Qatar.
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. CONCLUSION: 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.
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. CONCLUSION: 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.
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