Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool

Kojima, Keiichi; Miyoshi, Natsuki; Shibukawa, Atsushi; Chowdhury, Srikanta; Tsujimura, Masaki; Noji, Tomoyasu; Ishikita, Hiroshi; Yamanaka, Akihiro; Sudo, Yuki

doi:10.1021/acs.jpclett.0c01406

Cited by 18 publications

(36 citation statements)

References 19 publications

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“…Previously, we and others demonstrated that Cysl33, Serl56 and Tyr207 in Gt ACR1 (absorption maximum 515 nm) corresponding to Argl29, Glyl52 and Phe203 in Gt ACR2 (absorption maximum 470 nm) define the spectral difference between these two proteins (15, 26)(E.G. Govorunova, O.A.…”

Section: Resultsmentioning

confidence: 97%

Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution

Govorunova

Sineshchekov

et al. 2021

Preprint

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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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Section: Resultsmentioning

confidence: 97%

Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution

Govorunova

Sineshchekov

et al. 2021

Preprint

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“…To investigate whether the Om R2 gene encodes a photosensitive protein, we first expressed and purified its recombinant protein using the HEK293 cell expression system, which has been utilized for the functional expression of several eukaryotic rhodopsins from microbes and animal rhodopsins 29 , 38 . The purified Om R2 protein in detergent DDM micelles was colored purple and its absorption spectrum showed an absorption peak at 533 nm (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Nonetheless, we previously succeeded in expressing Cr ChR1 in E. coli cells by truncating the N- and C-termini of the proteins, although the truncated mutants showed constitutive activities that are different from the wild-type proteins 26 . We also succeeded in the functional expression and mutational analysis of Gt ACR2 in E. coli cells, which could provide a characteristic mutant for new optogenetics tools 27 – 29 . However, we were not able to purify the photoactive protein of Gt ACR2 from E. coli cells probably due to its denaturation during the solubilization step in detergent micelles.…”

Section: Introductionmentioning

confidence: 99%

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

Kikuchi

Kojima

Nakao

et al. 2021

Sci Rep

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Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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“…Protein expression and purification of GtACR1. The expression plasmids were transfected into HEK293T cells using the calcium-phosphate method 21,43 . After 1 day incubation, all-trans-retinal (final concentration = 5 μM) was added to transfected cells.…”

Section: Methodsmentioning

confidence: 99%

Proton-mediated gating mechanism of anion channelrhodopsin-1

Tsujimura

Kojima

Kawanishi

et al. 2021

Preprint

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Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths were also analyzed, using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shortens and lengthens the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients towards the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

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Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool

Cited by 18 publications

References 19 publications

Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution

Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

Proton-mediated gating mechanism of anion channelrhodopsin-1

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