Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function. Issue 3 (20th February 2020) - Seidel, Monika
Abstract: Aims : The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca 2+ 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 Ca 2+ -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 Ca 2+ 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. RyR2 R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca 2+ 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. Graphical Abstract: