The goal is to develop a detailed structural model for the cardiac ryanodine receptor (RyR)/calcium release channel that will contribute to understanding the mechanism of excitation- contraction coupling. The applicant will continue training in a combination of techniques: cryo-electron microscopy, single- particle image processing including three-dimensional reconstruction, and biochemical labeling. An initial preliminary three-dimensional reconstruction has been obtained recently, and work is in progress to improve resolution (expected resolution of 2-3 nm). Detailed comparisons will be made with existing reconstructions of the skeletal RyR which shows distinctive functional and sequence differences (66 percent identity) from the heart receptor. Reconstructions of the receptor under conditions that favor open and closed states will be determined, which will reveal quaternary and domain rearrangements that accompany channel gating, employing time-resolved (ms time scale) cryo-microscopy if necessary. Three-dimensional studies will begin of the RyR complexed with site-specific labels (e.g. sequence-specific antibodies, natural macromolecular modulators and ligands) that will allow nanometer-precision mapping of structural/functional sites on the RyR and provide important details on the architecture of the multicomponent signal transducing complex responsible for e-c coupling.
