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bioRxiv · 10.1101/2021.03.23.436664

Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution

Govorunova, E. G.·Sineshchekov, O. A.·Li, H.·Wang, Y.·Brown, L. S.·Palmateer, A.·Melkonian, M.·Cheng, S.·Carpenter, E.·Patterson, J.·Wong, G. K.-S.·Spudich, J. L.
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Abstract

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from three channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with the identical glutamate pattern as in cryptophyte ACRs are cation-selective. Desensitization is also broadly context-dependent, as in one branch of stramenopile ACRs and their metagenomic homologs its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, two prasinophyte CCRs exhibit red-shifted spectra to 585 nm, although their retinal-binding pockets do not match those of previously known similarly red-shifted channelrhodopsins. In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCEChannelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered as prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study we used manual and automated patch clamp recording of representative members of four channelrhodopsin families including a family that we report in this study in dinoflagellates. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

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Govorunova, E. G., Sineshchekov, O. A., Li, H., Wang, Y., Brown, L. S., Palmateer, A., Melkonian, M., Cheng, S., Carpenter, E., Patterson, J., Wong, G. K.-S., Spudich, J. L.. 2021-03-23. Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution. https://doi.org/10.1101/2021.03.23.436664

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