Natural Anion Channel Rhodopsins: A New Family of Tools for Optogenetic Neural Inhibition

Abstract

Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of efficient tools for membrane hyperpolarization. Anion Channel Rhodopsins (ACRs), natural light-gated anion channels recently discovered in cryptophyte algae [1], show distant homology to cation-conducting channelrhodopsins (CCRs), but strictly conduct anions. GtACR1 and GtACR2 from Guillardia theta hyperpolarize animal cell plasma membranes with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Their anion specificity, large currents, and fast kinetics enable ACRs to hyperpolarize and silence neurons through light-gated chloride conduction with unprecedented light sensitivity and temporal precision. The GtACRs are also the most conductive rhodopsin channels known, with 25-fold greater unitary conductance than the most commonly studied CCR, channelrhodopsin-2 [1]. Our initial findings on the gating mechanism of ACRs reveal that the structural basis for their anion conductance is fundamentally different from that of engineered chloride-conducting CCR mutants, not attributable to simply a modification of the CCR selectivity filter. During our analysis we identified an inverted mutant constitutively open in the dark and closed by light, the first such mutant of any light-gated channel, valuable for probing the structure of the channel open state. Also we identified a “step function” GtACR1 mutant useful in optogenetics [2].1. E.G. Govorunova, O.A. Sineshchekov, R. Janz, X. Liu, and J.L. Spudich (2015) Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics. Science 349:647-650.2. O.A. Sineshchekov, E.G. Govorunova, H. Li, and J.L. Spudich (2015) Gating mechanisms of a natural anion channelrhodopsin. Proc. Natl. Acad. Sci. USA, submitted.