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In channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle.

ABSTRACT: The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocycle is elucidated by time-resolved FTIR spectroscopy, which seems to be part of the opening mechanism of the conductive pore. Furthermore, Glu-90 is crucial to ion selectivity as also revealed by mutation of this residue combined with voltage clamp experiments. By dynamic homology modeling, we further hypothesized that the conductive pore is flanked by Glu-90 and located between helices A, B, C, and G.

SUBMITTER: Eisenhauer K

PROVIDER: S-EPMC3307317 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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In channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle.

Eisenhauer Kirstin K Kuhne Jens J Ritter Eglof E Berndt André A Wolf Steffen S Freier Erik E Bartl Franz F Hegemann Peter P Gerwert Klaus K

The Journal of biological chemistry 20120104 9

The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocy ...[more]

PMID: 22219197

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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

In channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle.

Publications

In channelrhodopsin-2 Glu-90 is crucial for ion selectivity and is deprotonated during the photocycle.

Similar Datasets

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