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Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation
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 functiona...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9700795/ https://www.ncbi.nlm.nih.gov/pubmed/36433995 http://dx.doi.org/10.1038/s41467-022-35018-6 |
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author | Baidukova, Olga Oppermann, Johannes Kelterborn, Simon Fernandez Lahore, Rodrigo G. Schumacher, Dimitri Evers, Heide Kamrani, Yousef Yari Hegemann, Peter |
author_facet | Baidukova, Olga Oppermann, Johannes Kelterborn, Simon Fernandez Lahore, Rodrigo G. Schumacher, Dimitri Evers, Heide Kamrani, Yousef Yari Hegemann, Peter |
author_sort | Baidukova, Olga |
collection | PubMed |
description | 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 Ca(2+) 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. |
format | Online Article Text |
id | pubmed-9700795 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-97007952022-11-27 Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation Baidukova, Olga Oppermann, Johannes Kelterborn, Simon Fernandez Lahore, Rodrigo G. Schumacher, Dimitri Evers, Heide Kamrani, Yousef Yari Hegemann, Peter Nat Commun Article 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 Ca(2+) 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. Nature Publishing Group UK 2022-11-25 /pmc/articles/PMC9700795/ /pubmed/36433995 http://dx.doi.org/10.1038/s41467-022-35018-6 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Baidukova, Olga Oppermann, Johannes Kelterborn, Simon Fernandez Lahore, Rodrigo G. Schumacher, Dimitri Evers, Heide Kamrani, Yousef Yari Hegemann, Peter Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation |
title | Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation |
title_full | Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation |
title_fullStr | Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation |
title_full_unstemmed | Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation |
title_short | Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation |
title_sort | gating and ion selectivity of channelrhodopsins are critical for photo-activated orientation of chlamydomonas as shown by in vivo point mutation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9700795/ https://www.ncbi.nlm.nih.gov/pubmed/36433995 http://dx.doi.org/10.1038/s41467-022-35018-6 |
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