Abstract 574: Protein-Protein Interactions between Components of the Cardiac Ryanodine Receptor Complex and their Impairment by Genetic Defects Linked to Sudden Cardiac Death
Intracellular Ca release in cardiac muscle is mediated by a mutimolecular complex composed among other proteins of the ryanodine receptor (RyR) channel, the integral proteins triadin (TRD) and junctin and the Ca binding proteins calsequestrin (CASQ2), which is also known to form polymers required for high capacity-intrastore Ca binding. Mutations of these proteins result in disregulated myocyte SR Ca release, cardiac arrhythmia and sudden death. We used fluorescence resonance energy transfer (FRET) and fusion constructs utilizing the cyan and yellow fluorescent proteins (CFP and YFP, respectively) as donor-acceptor pairs to investigate interactions between TRD and CASQ2 and between CASQ2 monomers in a heterologous expression system (3T3 cells). All fusion constructs were localized to the stores of the endoplasmic reticulum. When coexpressed, CFP-TRD and YFP-CASQ2 exhibited FRET the efficiency of which depended on the Ca loading state of the intracellular Ca stores. FRET efficiency was high when the stores were depleted and low when stores were Ca loaded, thus revealing a negative dependency of TRD-CASQ2 interaction on [Ca]. Cotransfection of CFP-CASQ2 and YFP-CASQ2 resulted in FRET with an efficiency that increased at high Ca store loads and decreased at low store loads, consistent with positive dependence of CASQ2-mulimerization. Expression of two different CASQ2 mutants (R33Q and G112+5X) linked to sudden cardiac death specifically affected the ability of CASQ2 either to interact with TRD or to polymerize. These results indicate that functionally- relevant interactions exists between components of the multimolecular RyR Ca release channel complex. These interactions could provide a molecular basis for the dynamic control of SR Ca release by intra-store Ca and for defective SR Ca release caused by mutations of CASQ2 to cause CPVT.