PROJECT SUMMARY/ABSTRACT: Ryanodine receptor-mediated calcium release plays an essential role in muscle contraction, and disruption of calcium homeostasis in excitable tissues caused by RyR-mediated calcium leak causes several genetic diseases, as well as contributing to the progressive loss of muscle function that occurs with aging. RyRs are tetrameric ion channels of unusually large size, with each subunit bearing a large cytosolic region that acts as a scaffold for the binding of allosteric modulators, which regulate the channel by binding at sites far from the transmembrane pore. The mechanistic basis of long-range allosteric modulation of RyR1 activity is not well understood. Understanding how allosteric regulators modulate RyR1 will both inform our understanding of RyR in a physiological context, and facilitate the development of molecules that target the channel to treat RyR1 related diseases. The goal of our proposal is to understand the structural mechanism of action of RyR1 allosteric modulators using single particle cryogenic electron microscopy (cryo-EM), complemented by functional analysis. We will investigate calmodulin and dantrolene as examples of long-range allosteric modulators which are endogenous protein binding partners and small molecule ligands, respectively. We will tackle this goal from three directions. In Aim 1, we seek to obtain a complete atomic model of RyR1, including of the peripheral domains where allosteric modulators bind, which have largely eluded sequence assignment due to their flexibility and mobility. We will apply a combination of symmetry expansion and masked refinement to generate a combined reconstruction with improved local resolution in peripheral regions, as well as explore optimization of sample preparation to obtain a more homogeneous particle set. In Aim 2, we will obtain structures of RyR1 in multiple functional states in complex with calmodulin and apocalmodulin, and to use single channel recordings of mutants of both calmodulin and RyR1 to test hypotheses arising from these structures. In Aim 3, we will investigate the mechanism of allosteric inhibition of RyR1 by dantrolene, by first structurally identifying the dantrolene binding site, and then examining the interdependence between inhibition by dantrolene and regulation by calmodulin, magnesium and ATP. Our research will broadly impact the field by unraveling the structural basis of allosteric regulation of an essential ion channel, RyR1. Structural characterization of the dantrolene binding site may lead the way to structure-based design of new RyR1 targeting therapeutics for the treatment of malignant hyperthermia and RyR1-related myopathies.