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Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin Chrimson.

ABSTRACT: Channelrhodopsins are light-gated ion channels of green algae used for the precise temporal and spatial control of transmembrane ion fluxes. The channelrhodopsin Chrimson from Chlamydomonas noctigama allows unprecedented deep tissue penetration due to peak absorption at 590?nm. We demonstrate by electrophysiological recordings and imaging techniques that Chrimson is highly proton selective causing intracellular acidification in HEK cells that is responsible for slow photocurrent decline during prolonged illumination. We localized molecular determinants of both high proton selectivity and red light activation to the extracellular pore. Whereas exchange of Glu143 only drops proton conductance and generates an operational Na-channel with 590?nm activation, exchange of Glu139 in addition increased the open state lifetime and shifted the absorption hypsochromic by 70?nm. In conjunction with Glu300 in the center and Glu124 and Glu125 at the intracellular end of the pore, Glu139 contributes to a delocalized activation gate and stabilizes by long-range interaction counterion configuration involving protonation of Glu165 that we identified as a key determinant of the large opsin shift in Chrimson.

SUBMITTER: Vierock J

PROVIDER: S-EPMC5577340 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin Chrimson.

Vierock Johannes J Grimm Christiane C Nitzan Noam N Hegemann Peter P

Scientific reports 20170830 1

Channelrhodopsins are light-gated ion channels of green algae used for the precise temporal and spatial control of transmembrane ion fluxes. The channelrhodopsin Chrimson from Chlamydomonas noctigama allows unprecedented deep tissue penetration due to peak absorption at 590 nm. We demonstrate by electrophysiological recordings and imaging techniques that Chrimson is highly proton selective causing intracellular acidification in HEK cells that is responsible for slow photocurrent decline during p ...[more]

PMID: 28855540

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Project description: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.

Dataset Information

Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin Chrimson.

Publications

Molecular determinants of proton selectivity and gating in the red-light activated channelrhodopsin Chrimson.

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