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

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.

Multifactorial in vivo regulation of the photoreceptor channelrhodopsin‐1 abundance

“…Integration would also be possible by feedback loops regulating the ChRs sensitivity and abundance using shared signaling elements. For the sensitivity regulation of the ChRs their conductivity for Ca 2+ and H + is central (Baidukova et al, 2022). A Ca 2+ -based feedback loop drives fast changes in the ChR1 phosphorylation status that are accompanied by changes in phototactic behavior.…”

“…The phototactic response is finally achieved by inducing subtle changes in the beating plane and frequency of the two cilia. These changes are the result of a yet largely unknown Ca 2+ ‐mediated signaling cascade initiated by the excitation of channelrhodopsins ChR1 and ChR2 toward the cilia (Baidukova et al, 2022; Kreimer et al, 2023). Both ChRs localize to the plasma membrane part of the eyespot (for review see Böhm & Kreimer [2021]).…”

“…Although a very detailed knowledge about ChRs in ectopic expression systems has accumulated in recent years, especially due to their central role in optogenetic studies (Deisseroth & Hegemann, 2017; Govorunova et al, 2017; Vierock & Hegemann, 2023), our knowledge about the in alga regulation of the ChRs is still at its infancy. Exceptions are the well‐studied fast photoreceptor currents evoked by their excitation (Baidukova et al, 2022; Kreimer et al, 2023; Sineshchekov et al, 2009). Further, phosphorylation is suggested to affect the ChR1 sensitivity.…”

Aberrant light sensing and motility in the green algaChlamydomonas priscuiifrom the ice-covered Antarctic Lake Bonney

“…To test for flagellar defects, we tested the photoshock response in C. priscuii . This ChR-mediated response occurs when algae sense a sudden and strong illumination, which causes a brief stop (<0.5s) and/or a period of backward motion (Baidukova et al ., 2022). It has been documented that the C. reinhardtii mutants ptx1 and lsp1 are not phototactic due to flagellar defects but display clear photoshock response as a result of a functional light-sensing and signalling apparatus (Okita et al ., 2005).…”

“…Our results suggest that C. priscuii encodes a single channelrhodopsin gene (ChR1), in contrast to two genes in the C. reinhardtii genome. The ChR photoreceptors are light-gated cation channels (Berthold et al, 2008), and are key determinants of phototactic responses in C. reinhardtii (Trippens et al, 2012;Greiner et al, 2017;Böhm et al, 2019;Wakabayashi et al, 2021;Baidukova et al, 2022). The sites for retinal binding, which are involved in light sensing and protein conformational change (Kato et al, 2012) Furthermore, none of the conserved phosphosites were in the C-terminal region of the C. priscuii ChR, which is of key importance for light-induced ChR1 hyperphosphorylation in C. reinhardtii (Böhm et al, 2019).…”