The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity

Govorunova, Elena G.; Sineshchekov, Oleg A.; Rodarte, Elsa M.; Janz, Roger; Morelle, Olivier; Melkonian, Michael; Wong, Gane Ka‐Shu; Spudich, John L.

doi:10.1038/srep43358

Cited by 99 publications

(94 citation statements)

References 45 publications

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“…Opsins with faster kinetics will extend the range of temporal patterns that can be produced in Purkinje cells (Govorunova et al, 2017; Gunayadin et al, 2010; Klapoetke et al, 2014; Lin et al, 2009). Devices that deliver spatially patterned lights (Farah et al, 2015; Zorzos et al, 2012) will broaden the range of useful manipulations further.…”

Section: Discussionmentioning

confidence: 99%

Selective Optogenetic Control of Purkinje Cells in Monkey Cerebellum

El-Shamayleh

Kojima

Soetedjo

et al. 2017

Neuron

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SUMMARY Purkinje cells of the primate cerebellum play critical but poorly understood roles in the execution of coordinated, accurate movements. Elucidating these roles has been hampered by a lack of techniques for manipulating spiking activity in these cells selectively—a problem common to most cell types in non-transgenic animals. To overcome this obstacle, we constructed AAV vectors carrying the channelrhodopsin-2 (ChR2) gene under the control of a 1 kb L7/Pcp2 promoter. We injected these vectors into the cerebellar cortex of rhesus macaques and tested vector efficacy in three ways. Immunohistochemical analyses confirmed selective ChR2 expression in Purkinje cells. Neurophysiological recordings confirmed robust optogenetic activation. Optical stimulation of the oculomotor vermis caused saccade dysmetria. Our results demonstrate the utility of AAV–L7–ChR2 for revealing the contributions of Purkinje cells to circuit function and behavior, and they attest to the feasibility of promoter-based, targeted, genetic manipulations in primates.

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Section: Discussionmentioning

confidence: 99%

Selective Optogenetic Control of Purkinje Cells in Monkey Cerebellum

El-Shamayleh

Kojima

Soetedjo

et al. 2017

Neuron

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“…5,10,20 In contrast, a heterogeneous chromophore configuration consisting of a mixture of all- trans - and 13- cis -retinal has been found to be present in Cr ChR2, which does undergo light–dark adaptation. 20,32,33 In general, ACRs similar to CCRs exhibit diverse properties such as kinetics, conductance, and spectral sensitivity, 34 and thus, it is not possible to generalize about their chromophore and protein structures, even in the unphotolyzed state, without detailed examination of individual ChRs.…”

Section: Discussionmentioning

confidence: 99%

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

Hai

Mamaeva

et al. 2017

Biochemistry

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A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel “open” state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet–visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational bands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid C=O stretching region indicate that several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 → Gln and Ser97 → Glu substitutions, residues located close to the retinylidene Schiff base, altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 → Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.

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“…A natural CCR variant with peak absorption in the orange-red spectral region has been discovered and used for photostimulation of neuronal firing (12). However, this CCR (named Chrimson) could not be converted to an anion channel by mutagenesis (13), and no natural ACRs with the absorption maximum beyond 540 nm had been found so far, despite extensive screening of homologous proteins from various cryptophyte species (14,15). Here we report that among the labyrinthulomycete ("Laby") ACRs), are four (we name "RubyACRs") that exhibit maximal spectral sensitivity in the orange-red region of the spectrum, dependent on unique residues of their retinal binding pocket.…”

Section: Introductionmentioning

confidence: 99%

RubyACRs, non-algal anion channelrhodopsins with highly red-shifted absorption

Govorunova

Sineshchekov

et al. 2020

Preprint

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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 labyrinthulomycetes and the other from haptophyte algae. Four closely related labyrinthulomycete ACRs, named RubyACRs here, exhibit a unique retinal binding pocket that creates spectral sensitivities with maxima at 590-610 nm, the most red-shifted channelrhodopsins known, long-sought for optogenetics, and more broadly the most red-shifted microbial rhodopsins so 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 labyrinthulomycete family. We also report energy transfer from a cytoplasmic fluorescent protein domain to the retinal chromophore bound within RubyACRs, not seen in similar constructs in other channelrhodopsins. Significance StatementOur identification and characterization of two ACR families, one from non-photosynthetic microorganisms, shows that light-gated anion conductance is more widely spread among eukaryotic lineages than previously thought. The uniquely far red-shifted absorption spectra of the subset we designate RubyACRs provide the long-sought inhibitory optogenetic tools producing large passive currents activated by long-wavelength light, enabling deep tissue penetration. Previously only low-efficiency ion-pumping rhodopsins were available for neural inhibition by the orange-red region of the spectrum. The unusual amino acid composition of the retinal-binding pocket in RubyACRs expands our understanding of color tuning in retinylidene proteins. Finally, energy transfer from the fluorescent protein used as a tag on RubyACRs opens a potential new dimension in molecular engineering of optogenetic tools. Main TextRecently a distinct family of ACRs was identified in environmental DNA samples of unknown organismal origin collected by the Tara Oceans project (7). This finding raised a possibility that channelrhodopsin genes were not limited to algae and were more widespread among eukaryotic protists. Here we report two distinct ACR families from labyrinthulomycetes and haptophytes. Labyrinthulomycetes are a class of aquatic saprotrophic microbes included in a larger group called stramenopiles (8). Labyrinthulomycete ancestors are believed to have never been photosynthetic (9), unlike some other heterotrophic eukaryotic lineages that evolved by plastid loss (e.g.

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The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity

Cited by 99 publications

References 45 publications

Selective Optogenetic Control of Purkinje Cells in Monkey Cerebellum

Selective Optogenetic Control of Purkinje Cells in Monkey Cerebellum

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

RubyACRs, non-algal anion channelrhodopsins with highly red-shifted absorption

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