Oligomerisation of the Human Cardiac Ryanodine Receptor Amino-Terminus

Abstract

The cardiac ryanodine receptor (RyR2) mediates the release of calcium from the sarcoplasmic reticulum of cardiac myocytes. The functional channel is composed of four identical subunits with the C-terminal part comprising the transmembrane domain predicted to form the Ca2+-conducting pore. The large N-terminal cytoplasmic portion of RyR2 is believed to serve as a scaffold for interaction with accessory proteins, ions and other regulatory molecules. RyR2 channel gating is regulated by a complex network of inter- and intra-subunit interactions between discrete structural domains. It has been proposed that disruption of inter-domain cross-talk results in abnormal RyR2 channel function, as observed in catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure.Here, we report that the RyR2 amino-terminus, containing one of the three CPVT-associated mutation hot spots, is capable of self-association. Chemical cross-linking of an RyR2 N-terminal fragment (BT4L; residues 1-906) indicated that it can assemble into tetramers. Moreover, BT4L expressed in mammalian HEK293 cells was found to form tetramers through endogenous disulphide bonds. We undertook a site-directed mutagenesis approach to identify the cysteines involved in BT4L disulphide bond formation, whereby cysteine residues were substituted by serine. The BT4L Cys361 Ser mutant did not form DTT-sensitive tetramers, suggesting that Cys361 participates in disulphide bond formation. When BT4L was co-expressed with full-length RyR2 in HEK293 cells it translocated from the cytosol to the microsomal fraction. The functional significance of RyR2 N-terminus self-association was studied by [3H]ryanodine binding assays. We found that the BT4L fragment activates the channel at low Ca2+ concentrations most likely by disrupting inter-subunit N-terminal self-association within the tetrameric channel. Our findings suggest that the RyR2 N-terminus regulates channel function through inter-subunit interactions. This work was supported by the British Heart Foundation.