Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation

Olga Baidukova (), Johannes Oppermann, Simon Kelterborn, Rodrigo G. Fernandez Lahore, Dimitri Schumacher, Heide Evers, Yousef Yari Kamrani and Peter Hegemann
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Olga Baidukova: Humboldt University of Berlin
Johannes Oppermann: Humboldt University of Berlin
Simon Kelterborn: Humboldt University of Berlin
Rodrigo G. Fernandez Lahore: Humboldt University of Berlin
Dimitri Schumacher: Humboldt University of Berlin
Heide Evers: Humboldt University of Berlin
Yousef Yari Kamrani: Humboldt University of Berlin
Peter Hegemann: Humboldt University of Berlin

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: Abstract The green unicellular alga Chlamydomonas reinhardtii with two photoreceptors called channelrhodopsins is a model organism that gave birth to a new scientific field of biomedical studies, optogenetics. Although channelrhodopsins are helping to decipher the activity of the human brain, their functionality has never been extensively studied in the organism of origin, mainly due to the difficulties connected to reverse genetic interventions. In this study, we present a CRISPR-Cas9-based technique that enables a precise in vivo exchange of single amino acids in a selected gene. To shed light on the function of channelrhodopsins ChR1 (C1) and ChR2 (C2) in vivo, we deleted both channelrhodopsins independently in the wild-type strain and introduced point mutations in the remaining channel, causing modified photocycle kinetics and ion selectivity. The mutated strains, ΔC1C2-E123T, ΔC1C2-E90R and ΔC1C2-E90Q, showed about 100-fold decrease in photosensitivity, a reduced photophobic response and faster light adaptation rates due to accelerated photocycle kinetics and reduced Ca2+ conductance. Moreover, the ΔC1C2-E90Q with an additionally reduced H+ permeability produced an electrical response only in the presence of Na+ ions, highlighting a contribution and importance of H+ conductance to photocurrents in the wild-type algae. Finally, in the ΔC1C2-E90R strain with the channelrhodopsin selectivity converted to anions, no photo-responses were detected. We conclude that the precise photocycle kinetics and the particular ion selectivity of channelrhodopsins are the key parameters for efficient phototaxis in low light conditions.

Date: 2022
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DOI: 10.1038/s41467-022-35018-6

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