Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications

Dawydow, Alexej; Gueta, Ronnie; Ljaschenko, Dmitrij; Ullrich, Sybille; Hermann, Moritz; Ehmann, Nadine; Gao, Shiqiang; Fiala, André; Langenhan, Tobias; Nagel, Georg; Kittel, Robert J.

doi:10.1073/pnas.1408269111

Cited by 178 publications

(244 citation statements)

References 45 publications

Supporting

Mentioning

235

Contrasting

Order By: Relevance

“…In particular, because the closing of the channel is influenced to a large extent, a larger proportion of the channels accumulate ChR in the open state ( Supplementary Fig. 2a,b), causing higher photosensitivity of DMAR-supplemented cells, as previously reported for step function rhodopsins 14,41,42,57 .…”

Section: Resultssupporting

confidence: 60%

See 1 more Smart Citation

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

et al. 2014

View full text Add to dashboard Cite

Optogenetic tools have become indispensable in neuroscience to stimulate or inhibit excitable cells by light. Channelrhodopsin-2 (ChR2) variants have been established by mutating the opsin backbone or by mining related algal genomes. As an alternative strategy, we surveyed synthetic retinal analogues combined with microbial rhodopsins for functional and spectral properties, capitalizing on assays in C. elegans, HEK cells and larval Drosophila. Compared with all-trans retinal (ATR), Dimethylamino-retinal (DMAR) shifts the action spectra maxima of ChR2 variants H134R and H134R/T159C from 480 to 520 nm. Moreover, DMAR decelerates the photocycle of ChR2(H134R) and (H134R/T159C), thereby reducing the light intensity required for persistent channel activation. In hyperpolarizing archaerhodopsin-3 and Mac, naphthyl-retinal and thiophene-retinal support activity alike ATR, yet at altered peak wavelengths. Our experiments enable applications of retinal analogues in colour tuning and altering photocycle characteristics of optogenetic tools, thereby increasing the operational light sensitivity of existing cell lines or transgenic animals.

show abstract

Section: Resultssupporting

confidence: 60%

“…However, the problem of most ChR variants being rather inefficient in the central nervous system of adult Drosophila could not be overcome. A recent ChR variant (ChR-XXL) with good expression and extremely long photocycle provides a very promising solution for this problem 57 .…”

Section: Discussionmentioning

confidence: 99%

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

et al. 2014

View full text Add to dashboard Cite

show abstract

“…This result has been attributed to a disruption of the hydrogen bond ("DC gate") that Cys-128 forms with Asp-156 in CrChR2 (12). Indeed, mutation of Asp-156 yielded comparable or even greater extension of the channel open time, as did that of Cys-128 (13,22). In GtACR1, the position of Asp-156 is occupied by Ser-130, which, according to our homology model, forms a hydrogen bond with Cys-102.…”

Section: Discussionmentioning

confidence: 99%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

102

View full text Add to dashboard Cite

Anion channelrhodopsins (ACRs) are a class of light-gated channels recently identified in cryptophyte algae that provide unprecedented fast and powerful hyperpolarizing tools for optogenetics. Analysis of photocurrents generated by Guillardia theta ACR 1 (GtACR1) and its mutants in response to laser flashes showed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH and involving different amino acid residues. The first mechanism, characterized by slow opening and fast closing of the channel, is regulated by Glu-68. Neutralization of this residue (the E68Q mutation) specifically suppressed this first mechanism, but did not eliminate it completely at high pH. Our data indicate the involvement of another, yetunidentified pH-sensitive group X. Introducing a positive charge at the Glu-68 site (the E68R mutation) inverted the channel gating so that it was open in the dark and closed in the light, without altering its ion selectivity. The second mechanism, characterized by fast opening and slow closing of the channel, was not substantially affected by the E68Q mutation, but was controlled by Cys-102. The C102A mutation reduced the rate of channel closing by the second mechanism by ∼100-fold, whereas it had only a twofold effect on the rate of the first. The results show that anion conductance by ACRs has a fundamentally different structural basis than the relatively well studied conductance by cation channelrhodopsins (CCRs), not attributable to simply a modification of the CCR selectivity filter.ion transport | channel gating | channelrhodopsins | optogenetics R ecently we reported a class of rhodopsins from the cryptophyte alga Guillardia theta that act as anion-conducting channels when expressed in cultured animal cells and therefore can be used to suppress neuronal firing by light (1). These proteinsnamed anion channelrhodopsins (ACRs)-show distant sequence homology to cation channelrhodopsins (CCRs) from chlorophyte (green) algae (2), but completely lack permeability for protons and metal cations. This property, as well as large current amplitudes and fast kinetics, makes ACRs superior hyperpolarizing optogenetic tools, compared with proton and chloride pumps (3, 4), or engineered Cl − -conducting CCR variants (5, 6). Two G. theta (Gt) ACRs differ in their spectral sensitivity and channel kinetics (1). GtACR1, with its absorption peak at 515 nm (Fig. S1), has an advantage over a more blue-shifted GtACR2 with maximal efficiency at 470 nm, because it allows using light of longer wavelengths that is less scattered by biological tissue.Because a CCR could be altered to exhibit Cl − channel activity by mutation of a single amino acid residue (5), a fundamental question about ACRs is whether they differ from CCRs only by their selectivity filter. In our search for an answer, we analyzed photocurrents generated by GtACR1 in HEK293 cells under single-turnover conditions in which secondary photochemistry does not complicate the photocycle. We found that GtACR1 cond...

show abstract

“…Although we used a cooling element, there was a slight increase in temperature (,1°) at the level of the arena due to the LED illumination. As published previously, all-trans-retinal (200 mM) was added to the standard medium to counter limited cellular availability of all-trans-retinal and thus increase efficiency of ChR2 expression for the UAS-ChR2-wt lines, but not for UAS-ChR2-XXL (Schroll et al 2006;Ullrich et al 2013;Dawydow et al 2014).…”

Section: Behavioral Assaysmentioning

confidence: 99%

Potency of Transgenic Effectors for Neurogenetic Manipulation inDrosophilaLarvae

et al. 2014

View full text Add to dashboard Cite

Genetic manipulations of neuronal activity are a cornerstone of studies aimed to identify the functional impact of defined neurons for animal behavior. With its small nervous system, rapid life cycle, and genetic amenability, the fruit fly Drosophila melanogaster provides an attractive model system to study neuronal circuit function. In the past two decades, a large repertoire of elegant genetic tools has been developed to manipulate and study neural circuits in the fruit fly. Current techniques allow genetic ablation, constitutive silencing, or hyperactivation of neuronal activity and also include conditional thermogenetic or optogenetic activation or inhibition. As for all genetic techniques, the choice of the proper transgenic tool is essential for behavioral studies. Potency and impact of effectors may vary in distinct neuron types or distinct types of behavior. We here systematically test genetic effectors for their potency to alter the behavior of Drosophila larvae, using two distinct behavioral paradigms: general locomotor activity and directed, visually guided navigation. Our results show largely similar but not equal effects with different effector lines in both assays. Interestingly, differences in the magnitude of induced behavioral alterations between different effector lines remain largely consistent between the two behavioral assays. The observed potencies of the effector lines in aminergic and cholinergic neurons assessed here may help researchers to choose the best-suited genetic tools to dissect neuronal networks underlying the behavior of larval fruit flies.

show abstract

Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications

Cited by 178 publications

References 45 publications

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

Gating mechanisms of a natural anion channelrhodopsin

Potency of Transgenic Effectors for Neurogenetic Manipulation inDrosophilaLarvae

Contact Info

Product

Resources

About