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Proc Natl Acad Sci U S A. 2016 Jan 26;113(4):822-9. doi: 10.1073/pnas.1523341113. Epub 2015 Dec 22.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

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Department of Bioengineering, Stanford University, Stanford, CA 94305;
Institute for Biology, Experimental Biophysics, Humboldt Universität zu Berlin, D-10115 Berlin, Germany;
Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA 94305; Nancy Pritzker Laboratory, Stanford University, Stanford, CA 94305;
Program in Neurosciences and Mental Health, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada M5G 1X8;
Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden;
Department of Bioengineering, Stanford University, Stanford, CA 94305; Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA 94305; Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305


The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.


channelrhodopsin; chloride; neuronal inhibition; optogenetics; structure

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