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RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption.

ABSTRACT: Channelrhodopsins are light-gated ion channels widely used to control neuronal firing with light (optogenetics). We report two previously unknown families of anion channelrhodopsins (ACRs), one from the heterotrophic protists labyrinthulea and the other from haptophyte algae. Four closely related labyrinthulea ACRs, named RubyACRs here, exhibit a unique retinal-binding pocket that creates spectral sensitivities with maxima at 590 to 610 nm, the most red-shifted channelrhodopsins known, long-sought for optogenetics, and more broadly the most red-shifted microbial rhodopsins thus far reported. We identified three spectral tuning residues critical for the red-shifted absorption. Photocurrents recorded from the RubyACR from Aurantiochytrium limacinum (designated AlACR1) under single-turnover excitation exhibited biphasic decay, the rate of which was only weakly voltage dependent, in contrast to that in previously characterized cryptophyte ACRs, indicating differences in channel gating mechanisms between the two ACR families. Moreover, in A. limacinum we identified three ACRs with absorption maxima at 485, 545, and 590 nm, indicating color-sensitive photosensing with blue, green, and red spectral variation of ACRs within individual species of the labyrinthulea family. We also report functional energy transfer from a cytoplasmic fluorescent protein domain to the retinal chromophore bound within RubyACRs.

SUBMITTER: Govorunova EG

PROVIDER: S-EPMC7502701 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption.

Govorunova Elena G EG Sineshchekov Oleg A OA Li Hai H Wang Yumei Y Brown Leonid S LS Spudich John L JL

Proceedings of the National Academy of Sciences of the United States of America 20200901 37

Channelrhodopsins are light-gated ion channels widely used to control neuronal firing with light (optogenetics). We report two previously unknown families of anion channelrhodopsins (ACRs), one from the heterotrophic protists labyrinthulea and the other from haptophyte algae. Four closely related labyrinthulea ACRs, named RubyACRs here, exhibit a unique retinal-binding pocket that creates spectral sensitivities with maxima at 590 to 610 nm, the most red-shifted channelrhodopsins known, long-soug ...[more]

PMID: 32873643

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Project description:When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM), this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

Project description:BackgroundHuman African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.Methodology/principal findingsHere, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5'-variant surface glycoprotein untranslated region (UTR) and a downstream 3'-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.Conclusions/significanceThe optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.

Project description: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 four 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 a glutamate pattern identical to that 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, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). 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. IMPORTANCE Channelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered 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 in dinoflagellates that we report in this study. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

Dataset Information

RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption.

Publications

RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption.

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