SSA - The in situ molecular structure of active calcium ion channels

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Background:Structural mechanisms involving calcium channels underpin various neurological functions including sensation and the acquisition of memory. Our recent published research indicates that Ca2+ signals are not propagated by single channel complexes. Instead, higher order macromolecular supercomplexes act in concert to coordinate local structural remodeling within the cell, includingsynapse enlargement by actin polymerization and the recruitment of smooth ER to the synaptic membrane - the structural basis for these active cellular processes is currently unknown. We have developed methods for investigating the in situ molecular structure of synapses by cryogenic correlated light-electron microscopy, cryo-electron tomography and sub-tomogram averaging. Thus, our goal is to understand the structural basis and molecular mechanism of Ca+2 - dependent signal transduction at the synaptic membrane.Novelty and timeliness:Cryo-electron microscopy (cryoEM) is the fastest growing discipline in structural biology, where advancements in instrumentation and computational image analysis are transforming our understanding of molecular mechanisms. With its investment in cryoEM Leeds is at the forefront of this revolution. We expect that by combining mouse genetics with cryoEM to determine in situmolecular structures within cells and tissues will represent a further significant advancement in this field. Experimental approach:We are combining mouse genetics, pharmacological fluorescent tags, correlated light-electron microscopy, and cryo-electron tomography to locate ion channel complexes within tissue and cell samples to determine in situ molecular structure. The mechanism of activity-dependent structural change within cells and tissues will be investigated using fluorescent calcium indicators to locateactive membranes for structure determination.