New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>

ABSTRACT Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rho...

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Autores principales:	Elena G. Govorunova, Elena N. Spudich, C. Elizabeth Lane, Oleg A. Sineshchekov, John L. Spudich
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2011
Materias:	Microbiology QR1-502
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spelling	oai:doaj.org-article:a0b7e0ef8b354127a615c7c3ea2fe7fe2021-11-15T15:38:49ZNew Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>10.1128/mBio.00115-112150-7511https://doaj.org/article/a0b7e0ef8b354127a615c7c3ea2fe7fe2011-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00115-11https://doaj.org/toc/2150-7511ABSTRACT Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name “channelrhodopsins.” Channelrhodopsins have become useful molecular tools for light control of cellular activity. Only four channelrhodopsins, identified in Chlamydomonas reinhardtii and Volvox carteri, have been reported so far. By screening light-induced currents among algal species, we identified that the phylogenetically distant flagellate Mesostigma viride showed photoelectrical responses in vivo with properties suggesting a channelrhodopsin especially promising for optogenetic use. We cloned an M. viride channelrhodopsin, MChR1, and studied its channel activity upon heterologous expression. Action spectra in HEK293 cells match those of the photocurrents observed in M. viride cells. Comparison of the more divergent MChR1 sequence to the previously studied phylogenetically clustered homologs and study of several MChR1 mutants refine our understanding of the sequence determinants of channelrhodopsin function. We found that MChR1 has the most red-shifted and pH-independent spectral sensitivity so far reported, matches or surpasses known channelrhodopsins’ channel kinetics features, and undergoes minimal inactivation upon sustained illumination. This combination of properties makes MChR1 a promising candidate for optogenetic applications. IMPORTANCE Channelrhodopsins that function as phototaxis receptors in flagellate algae have recently come into the spotlight as genetically encoded single-molecule optical switches for turning on neuronal firing or other cellular processes, a technique called “optogenetics.” Only one of four currently known channelrhodopsins is widely used in optogenetics, although electrical currents recorded in diverse flagellates suggest the existence of a large variety of such proteins. We applied a strategy for the search for new channelrhodopsins with desirable characteristics by measuring rhodopsin-mediated photocurrents in microalgae, which helped us identify MChR1, a new member of the channelrhodopsin family. MChR1 exhibits several sought-after characteristics and thus expands the available optogenetic toolbox. The divergence of the MChR1 sequence from those of the four known channelrhodopsins contributes to our understanding of diversity in the primary structures of this subfamily of sensory rhodopsins.Elena G. GovorunovaElena N. SpudichC. Elizabeth LaneOleg A. SineshchekovJohn L. SpudichAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 2, Iss 3 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Elena G. Govorunova Elena N. Spudich C. Elizabeth Lane Oleg A. Sineshchekov John L. Spudich New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>
description	ABSTRACT Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name “channelrhodopsins.” Channelrhodopsins have become useful molecular tools for light control of cellular activity. Only four channelrhodopsins, identified in Chlamydomonas reinhardtii and Volvox carteri, have been reported so far. By screening light-induced currents among algal species, we identified that the phylogenetically distant flagellate Mesostigma viride showed photoelectrical responses in vivo with properties suggesting a channelrhodopsin especially promising for optogenetic use. We cloned an M. viride channelrhodopsin, MChR1, and studied its channel activity upon heterologous expression. Action spectra in HEK293 cells match those of the photocurrents observed in M. viride cells. Comparison of the more divergent MChR1 sequence to the previously studied phylogenetically clustered homologs and study of several MChR1 mutants refine our understanding of the sequence determinants of channelrhodopsin function. We found that MChR1 has the most red-shifted and pH-independent spectral sensitivity so far reported, matches or surpasses known channelrhodopsins’ channel kinetics features, and undergoes minimal inactivation upon sustained illumination. This combination of properties makes MChR1 a promising candidate for optogenetic applications. IMPORTANCE Channelrhodopsins that function as phototaxis receptors in flagellate algae have recently come into the spotlight as genetically encoded single-molecule optical switches for turning on neuronal firing or other cellular processes, a technique called “optogenetics.” Only one of four currently known channelrhodopsins is widely used in optogenetics, although electrical currents recorded in diverse flagellates suggest the existence of a large variety of such proteins. We applied a strategy for the search for new channelrhodopsins with desirable characteristics by measuring rhodopsin-mediated photocurrents in microalgae, which helped us identify MChR1, a new member of the channelrhodopsin family. MChR1 exhibits several sought-after characteristics and thus expands the available optogenetic toolbox. The divergence of the MChR1 sequence from those of the four known channelrhodopsins contributes to our understanding of diversity in the primary structures of this subfamily of sensory rhodopsins.
format	article
author	Elena G. Govorunova Elena N. Spudich C. Elizabeth Lane Oleg A. Sineshchekov John L. Spudich
author_facet	Elena G. Govorunova Elena N. Spudich C. Elizabeth Lane Oleg A. Sineshchekov John L. Spudich
author_sort	Elena G. Govorunova
title	New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>
title_short	New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>
title_full	New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>
title_fullStr	New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>
title_full_unstemmed	New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>
title_sort	new channelrhodopsin with a red-shifted spectrum and rapid kinetics from <italic toggle="yes">mesostigma viride</italic>
publisher	American Society for Microbiology
publishDate	2011
url	https://doaj.org/article/a0b7e0ef8b354127a615c7c3ea2fe7fe
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New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from <italic toggle="yes">Mesostigma viride</italic>

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