Structural and mechanistic basis of channelrhodopsin function

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.

Grant Number: 5R01GM148599-04
NIH Institute/Center: NIH
Principal Investigator: Stephen Brohawn