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

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.

Cardiac RyR N-terminal region biosensors for FRET-based high-throughput screening

“…Of the five validated hits, four shortened the FLT (increased FRET), which suggests a shorter inter-probe distance within NTR; and one lengthened FLT (decreased FRET), which suggests a longer inter-probe distance. All five FLT hits also affected [ 3 H]ryanodine binding to RyR1 and 2, which provides experimental support to the hypothesis that NTR ligands can regulate the functional status of RyR channels, as suggested by high-resolution structures of RyR channels and many functional studies (17,26,33).…”

“…Very recently, it was also proposed that NTR self-association is the "gatekeeper" of RyR2 channel activity. Specifically, a stable N-terminal tetramer maintains the RyR2 channel closure, whereas disruption of this tetramer results in channel dysfunction (33). In addition, the NTR is one of the three major clusters of mutations associated with skeletal and cardiac myopathies such as malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT) (26,27,(34)(35)(36).…”

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

“…Of the five validated hits, four shortened the FLT (increased FRET), suggesting a shorter interprobe distance within NTR; and one lengthened FLT (decreased FRET), suggesting a longer interprobe distance. All five FLT hits also affected [ 3 H]ryanodine binding to RyR1 and RyR2, which provides experimental support to the hypothesis that NTR ligands can regulate the functional status of RyR channels, as suggested by high-resolution structures of RyR channels and many functional studies ( 17 , 26 , 28 ). Although the FRET biosensor has been developed based on the RyR2-NTR, there is a high degree of homology with the RyR1-NTR (∼70% sequence identity).…”

“…Very recently, it was proposed that NTR self-association is the “gatekeeper” of RyR2 channel activity. Specifically, a stable N-terminal tetramer maintains the RyR2 channel closure, whereas disruption of this tetramer results in channel dysfunction ( 28 ). In addition to structural evidence, the NTR is one of the three major clusters of mutations associated with skeletal and cardiac myopathies such as malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT) ( 26 , 27 , 29 , 30 ).…”

Molecular, Subcellular, and Arrhythmogenic Mechanisms in Genetic RyR2 Disease

“…They are not making direct contact with the pore-forming region, but their effects are mediated via the CSol. Indeed, the NTD, Jsol, Bsol, and CSol are involved in multiple intra- and inter-subunit interactions with each other [ 9 , 10 , 18 , 19 , 20 ]. The NTD also interacts with itself to promote channel closure as well as to support the formation of functional tetrameric RyR2 channels [ 21 , 22 , 23 ].…”