Structural and mechanistic basis of channelrhodopsin function

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PROJECT SUMMARY The goal of this project is to understand the mechanistic basis for gating and function in channelrhodopsins, retinal-binding proteins that are similar to vertebrate visual proteins and form light-gated ion channels to control phototaxis in motile algae. In the nearly two decades since they were first cloned, channelrhodopsins have become important models for understanding membrane protein structure, function, and biophysics and widely utilized molecular tools in optogenetics, in which their heterologous expression in genetically targeted cells enables control of membrane potential and electrical excitability with light. Here, we will apply cryo-electron microscopy to determine structures of channelrhodopsins in different functional states and electrophysiological recordings of structure-based variants to understand the basis for channel gating and determinants for key channel properties. We aim to capture structural snapshots of different open and closed conformations by identifying combinations of stimulation conditions and channel variants that promote different states. We will leverage these structural insights to interrogate the molecular basis for diverse kinetics, conductance, and spectral sensitivity among channelrhodopsins and derive physical models for gating and functional properties. We will focus our efforts on two channelrhodopsins that are the most potent members of the two depolarizing channel families widely used in optogenetics, the cation channelrhodopsins (CCRs) and bacteriorhodopsin-like cation channelrhodopsins (BCCRs). CCRs and BCCRs share a common architecture, but are structurally, evolutionarily, and mechanistically distinct. Comparative analyses of these two channelrhodopsin families will therefore provide additional insight into how light energy is converted into gating conformational changes and the molecular basis for channel activity. Since the initial characterization and cloning of channelrhodopsins, the optogenetic toolbox has been greatly expanded by the engineering of novel channelrhodopsins with varied and improved properties. Still, these efforts have been limited to date by an incomplete understanding of the structural and mechanistic basis for channel function. Therefore, in addition to providing fundamental mechanistic insight into channelrhodopsin gating and activity, this work will serve as a basis for the rational design of new channelrhodopsin variants with modified properties that further expand the potential of optogenetic manipulations. Such tools could enable new experiments at larger scale, in deeper tissue, in larger organisms, and with higher precision. They could also lead to new clinical approaches for treating disease including those of the nervous and cardiovascular systems.