Rational design of optogenetic tools : from bioinformatic genomic data analysis to electrophysiological validation

Alekseev, Alexey; Gordeliy, Valentin (Thesis advisor); Büldt, Georg (Thesis advisor); Bamberg, Ernst (Thesis advisor); Engelhard, Martin (Thesis advisor)

Aachen : Rheinisch-Westfälische Technische Hochschule Aachen (2020, 2021)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020

Abstract

Microbial rhodopsins comprise one of the most diverse clades of light-harvesting proteins. At the beginning of the 21st century, they found exceptional application in neuroscience with the potential for the invention of medical therapies of the new generation. Currently, a variety of channelrhodopsins were functionally described. However, the search for optogenetic tools with desired properties is still a challenge. In the thesis, we describe a pipeline for the identification of microbial rhodopsins with potentially new properties and their structural and functional characterization. The pipeline is based on the method of bioinformatics, taking into account current knowledge of microbial rhodopsins and known x-ray structures, and patch-clamp recordings in mammalian cells and cultured neurons. First, we identified several different clades of rhodopsins type-1, which might have unique functional properties for optogenetics. Second, we conducted a detailed structure-based bioinformatic analysis of heliorhodopsins and showed that they form a heterogeneous class of proteins. We identified several groups of heliorhodopsins that may possess different functions. Third, we examined the electrophysiology of VirChR1, a member of viral rhodopsins group 1. VirChR1 turned out to be ion-channel selective for monovalent cations. Besides, we showed that it could fire cultured rat hippocampal neurons. We showed that the VirChR1 is impermeable to Ca2+, which makes this protein a potentially perspective tool for optogenetics. Taking into account all data, we conclude that viral rhodopsins and heliorhodopsins are widely distributed and also suggest their involvement in the global ecology.

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