Conversion of Channelrhodopsin into a Light-Gated Chloride Channel

Wietek, Jonas; Wiegert, J. Simon; Adeishvili, Nona; Schneider, Franziska; Watanabe, Hiroshi; Tsunoda, Satoshi P.; Vogt, Arend; Elstner, Marcus; Oertner, Thomas G.; Hegemann, Peter

doi:10.1126/science.1249375

Cited by 342 publications

(333 citation statements)

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“…An exchange from Cl − to SO 4 2− buffer caused a 24-nm red shift of the spectral maximum of purified halorhodopsin from Natronomonas pharaonis (12). In contrast, no spectral shift was detected upon the same substitution in GtACR1, which strongly argues against Cl − playing a role of the Schiff base counterion in the latter pigment.…”

Section: Discussionmentioning

confidence: 91%

“…It is also typical of halorhodopsins in which Cl − serves as a counterion to the protonated Schiff base (11). To test the possibility of Cl − acting as a Schiff base counterion in GtACR1, we made measurements in a deionized sample and a sample in which Cl − was replaced with SO 4 2− in the buffer. In contrast to halorhodopsin (12), no spectral changes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, imposing early M formation on GtACR1 by engineering a proton acceptor adjacent to the protonated Schiff base nearly completely inhibits channel opening, indicating that the presence of a noncarboxylate residue in the position of the canonical proton acceptor (Asp-85 in BR) is critical for ACR function. Deionization of purified pigment or substitution of SO 4 2− for Cl − in the buffer changed neither the position of the absorption maximum nor the photocycle, which argues against Cl − being a Schiff base counterion in GtACR1.…”

Section: Significancementioning

confidence: 90%

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Photochemical reaction cycle transitions during anion channelrhodopsin gating

Sineshchekov

Govorunova

et al. 2016

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

130

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A recently discovered family of natural anion channelrhodopsins (ACRs) have the highest conductance among channelrhodopsins and exhibit exclusive anion selectivity, which make them efficient inhibitory tools for optogenetics. We report analysis of flashinduced absorption changes in purified wild-type and mutant ACRs, and of photocurrents they generate in HEK293 cells. Contrary to cation channelrhodopsins (CCRs), the ion conducting state of ACRs develops in an L-like intermediate that precedes the deprotonation of the retinylidene Schiff base (i.e., formation of an M intermediate). Channel closing involves two mechanisms leading to depletion of the conducting L-like state: (i) Fast closing is caused by a reversible L⇔M conversion. Glu-68 in Guillardia theta ACR1 plays an important role in this transition, likely serving as a counterion and proton acceptor at least at high and neutral pH. Incomplete suppression of M formation in the GtACR1_E68Q mutant indicates the existence of an alternative proton acceptor. (ii) Slow closing of the channel parallels irreversible depletion of the M-like and, hence, L-like state. Mutation of Cys-102 that strongly affected slow channel closing slowed the photocycle to the same extent. The L and M intermediates were in equilibrium in C102A as in the WT. In the position of Glu-123 in channelrhodopsin-2, ACRs contain a noncarboxylate residue, the mutation of which to Glu produced early Schiff base proton transfer and strongly inhibited channel activity. The data reveal fundamental differences between natural ACR and CCR conductance mechanisms and their underlying photochemistry, further confirming that these proteins form distinct families of rhodopsin channels.photochemical conversions | Schiff base | channel gating | channelrhodopsins | optogenetics T he genomes of cryptophyte algae harbor nucleotide sequences that encode anion-conducting channelrhodopsins (ACRs) (1, 2). Although these proteins show distant sequence homology to cation-conducting channelrhodopsins (CCRs) from green (chlorophyte) algae, they completely lack permeability for protons and metal cations and, thus, represent a distinct structural and functional class among microbial rhodopsins. Using ACRs permits optogenetic inhibition of neuronal firing at much lower light intensities than other currently used silencers (1).Analysis of photocurrents generated by ACR1 from Guillardia theta (GtACR1) in human embryonic kidney (HEK) cells under single-turnover conditions revealed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH, and involving different amino acid residues (3). The first mechanism, characterized by fast closing of the channel, is regulated by Glu-68, a homolog of Glu-90 in channelrhodopsin-2 from the green alga Chlamydomonas reinhardtii (CrChR2). Replacement of Glu-90 with Arg in CrChR2 made the channel permeable for Cl − (4), whereas the same substitution of Glu-68 in GtACR1 did not influence its selectivity for anions, but inverted its gating, ren...

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Section: Discussionmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 90%

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Photochemical reaction cycle transitions during anion channelrhodopsin gating

Sineshchekov

Govorunova

et al. 2016

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

130

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“…According to the primary sequence alignment, Glu-68 corresponds to Glu-90, the residue that determines cation selectivity of CrChR2 (7,14,15). Mutation of Glu-90 to Lys or Arg converted CrChR2 to an anion channel with residual permeability for protons (5). However, the selectivity filter of ACRs appears to be different from that of the CrChR2_E90K/R mutants, because the presence of the Glu-90 homolog in WT GtACRs is obviously not a barrier to anion permeation.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

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

102

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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...

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Future focal treatment approaches to epilepsy

Ritter¹,

Cock²

2015

The Treatment of Epilepsy

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Conversion of Channelrhodopsin into a Light-Gated Chloride Channel

Cited by 342 publications

References 31 publications

Photochemical reaction cycle transitions during anion channelrhodopsin gating

Photochemical reaction cycle transitions during anion channelrhodopsin gating

Gating mechanisms of a natural anion channelrhodopsin

Future focal treatment approaches to epilepsy

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