Localization of the Dantrolene Target Sequence in the Cardiac Ryanodine Receptor by Combining cryo-EM and FRET Analysis

Abstract

Mutations in ryanodine receptors (RyRs) cause calcium channel dysfunction in two skeletal muscle diseases, malignant hyperthermia (MH) and central core disease, and in polymorphic ventricular tachycardia in cardiac muscle. MH is an inherited condition that causes muscle rigidity and uncontrollable fever in reaction to administration of certain anesthetics. Dantrolene is a clinical drug that suppresses spontaneous Ca2+ release and is used as a therapeutic agent for individuals susceptible to MH. Recently, dantrolene has been shown to improve cardiomyocyte function in failing hearts. Biochemical studies suggest that the N-terminal region of RyR (amino acids 590-609 in RyR1 and 601-620 in RyR2) is the molecular target for dantrolene. We have attempted to localize the dantrolene target sequence by 3D cryo-electron microscopy. However, inserting a GFP after Arg-626 in RyR2 abolished the receptors’ binding to the GST-FKBP12.6 affinity column and prevented the purification of RyR2R626-GFP for structural studies. As an alternative approach, we designed several fluorescence resonance energy transfer (FRET) pairs based on our previous cryo-EM structures to map the location of Arg-626. We generated four pairs of dual insertions in RyR2 (RyR2S437-YFP/R626-CFP, RyR2R626-CFP/Y846-YFP, RyR2S437-YFP/Y846-CFP, and RyR2R626-YFP/S2367-CFP); three pairs between AF555-FKBP12.6 and RyR2-GFP (RyR2S437-GFP, RyR2R626-GFP, and RyR2Y846-GFP); and three pairs between CFP-FKBP12.6 and RyR2-YFP (RyR2S437-YFP, RyR2R626-YFP, and RyR2Y846-YFP). We estimated the 3D location of dantrolene binding sequence by measuring the FRET efficiencies in each of these pairs, and by correlating FRET efficiencies to the distance between donor and acceptor with known locations. Our results reveal the dantrolene target sequence is likely to be located in the clamp region close to the FKBP12.6 binding site.