Defining architecture of EC coupling machinery in situ

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Project Summary/Abstract The focus of this proposal is on excitation-contraction coupling (ECC) in skeletal muscle. The ECC consists of a series of physiological events linking the depolarization of muscle cell’s plasma membrane to the release of Ca2+ from the sarcoplasmic reticulum (SR) into cytoplasm, resulting in muscle contraction. ECC is restricted spatially to a subcompartment of muscle cells (‘triad junction’) and regulated precisely via a physical interaction between the voltage-gated Ca2+ channel (dihydropyridine receptor, DHPR) on the plasma membrane and the Ca2+-release channel (type 1 ryanodine receptor, RyR1) in the SR. Many drugs currently in use to treat muscle disorders target these two Ca2+ channels. Despite recent remarkable advances in the structural characterization of these two channels, the molecular mechanisms underlying their interactions remain elusive due to the lack of detailed 3D architecture of the ECC machinery comprising both channels and associated regulatory proteins. Determining architecture of such multiprotein complexes is a formidable challenge given their native location in lipid membranes and the lack a general means to preserve the complex integrity upon extraction with detergents from their lipid bilayer environment. In this project, we will address this challenge by utilizing advanced cryogenic electron tomography (cryoET) to study frozen-hydrated triad junctions isolated from skeletal muscle (aim 1) as well as within myotubes cultured on EM grids (aim 2). To accomplish these studies, we endeavor to develop the experimental workflow for in situ cryoET analysis of the ECC complex. This workflow will consist of the following major steps: preparation of the membrane-embedded ECC complexes suitable for cryoET analysis; cryoET data collection, image analysis, tomographic reconstruction and subtomogram averaging; visualization and annotation of densities in cryo-tomograms. The determined structures will reveal mechanistically informative features underlying protein-protein interactions in the ECC Ca2+ release complex that will allow important functional insights into the ECC process. In the future, we will apply the workflow developed here to structure- functional characterization of ECC in different types of muscle and under pathological conditions. Overall, the proposed studies are highly significant, as they will provide mechanistic structural insights into the ECC machinery illuminating the pathological consequences of deregulated Ca2+ signaling, that will ultimately aid in search for novel therapies targeting neuromuscular diseases. The workflow developed, as part of this research will have broad applicability to studies of other integral membrane protein complexes.