Evolution of the Channelrhodopsin Photocycle Model

Stehfest, Katja; Hegemann, Peter

doi:10.1002/cphc.200900980

Cited by 64 publications

(83 citation statements)

References 42 publications

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“…In contrast, in CrChR2 two conducting states have been recognized by their different relative permeability to H + and Na + (23,24). A model of two interconnected photocycles, each of which contains a closed (nonconducting) state and an open (conducting) state, has been developed for CCRs (25)(26)(27). However, single-turnover photocurrents generated by CrChR2 upon laser excitation decay monoexponentially (28,29), which means that the second conducting state accumulates only under continuous light, indicating that the second state requires secondary photochemistry.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…41 Channelrhodopsin-2 from C. reinhardtii, CrChR2, is the best characterized ChR so far, both by electrophysiological and spectroscopic studies, as recently reviewed. 42,43 CrChR2 absorbs maximally at ∼470 nm and shows a photocycle comprising a minimum of four intermediate states: 43 The only ChR whose 3D structure has been resolved to atomic resolution is a chimera construct from CrChR1 and CrChR2 (C1C2). 44 Recently, the first report on active internal water molecules on a ChR was published, performed on the C1C2 chimera.…”

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confidence: 99%

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

Lórenz-Fonfrı́a

Muders

Schlesinger

et al. 2014

The Journal of Chemical Physics

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Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the ( −1 ) to moderately strongly (3300 cm −1 ) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1.

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“…Previous work on channelrhodopsins CrChR2 and PsChR2 indicated that laser-induced current transients roughly correlate with late photocycle intermediate(s) observed in independent photochemical studies (9,10). The complex time dependence of the current response to step or continuous illumination, however, is difficult to reconcile with the reaction sequence derived from photochemical measurements (9,(11)(12)(13)(14). The desire to explain the unusual * This work was supported by National Institutes of Health Grant R01EY000983 from the NEI (to D. S. K.).…”

Section: Channelrhodopsins (Chrs)mentioning

confidence: 96%

Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function

Szundi

Bogomolni

Kliger

2015

Journal of Biological Chemistry

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Background: Channelrhodopsins (ChRs) are light-gated cation channels widely used in optogenetics. Results: The photocycle obtained from time-resolved absorption spectroscopy of PsChR2 is shown to explain photocurrent kinetics observed in HEK293 cells. Conclusion:The photochemical reactions involving two parallel photocycles result in the complex photocurrent behavior. Significance: The parallel-cycle model opens new perspectives in understanding photocurrents from channelrhodopsins.

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Evolution of the Channelrhodopsin Photocycle Model

Cited by 64 publications

References 42 publications

Gating mechanisms of a natural anion channelrhodopsin

Gating mechanisms of a natural anion channelrhodopsin

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

Platymonas subcordiformis Channelrhodopsin-2 (PsChR2) Function

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