The DC gate in Channelrhodopsin-2: crucial hydrogen bonding interaction between C128 and D156

Nack, Melanie; Radu, Ionela; Gossing, Michael; Bamann, Christian; Bamberg, Ernst; Mollard, Gabriele Fischer von; Heberle, Joachim

doi:10.1039/b9pp00157c

Cited by 87 publications

(130 citation statements)

References 18 publications

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“…The effects of C128X mutations on channel activity have been best studied in CrChR2 (11, 13), but a similar dramatic decrease in the current decay rate was also observed in the corresponding mutants of other CCRs, such as Mesostigma viride channelrhodopsin 1 (MvChR1) (21). 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).…”

Section: Discussionmentioning

confidence: 93%

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: 93%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

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

102

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“…the neighboring residue of L94. In ChR2, the interaction of TM3 and TM4 seems to affect both gating and selectivity, pointing to a structural element as transducer of the light reaction to the ion pore 24 . Insertion of the smaller and more hydrophilic cysteine could increase the flexibility of the helical segment, facilitating the access of Ca ++ .…”

Section: Application To Hippocampal Neuronsmentioning

confidence: 99%

Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh

et al. 2011

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“…It is surprising that mutations of either C128 or D156 in ChR2 lead to far more drastic functional alterations with intensified photocurrents and strongly retarded ground-state recovery kinetics (7) because these residues are located in helices C and D, respectively (i.e., remote from the proposed cation channel). The interaction of C128 and D156 was shown by FTIR difference spectroscopy to involve an H-bond between the termini of both side chains, the so-called "DC gate" (8). The structural basis of the DC gate could not be corroborated by the crystallographic model of C1C2, where the terminal S-H of C128 points away from D156 (6).…”

mentioning

confidence: 96%

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating

Lórenz-Fonfrı́a

Resler

Krause

et al. 2013

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

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160

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The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P 2 390 and P 3 520 ) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t 1/2 = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t 1/2 = 10 μs), with the latter being reprotonated by aspartic acid 156 (t 1/2 = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P 4 480 state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.O ptogenetics provides new tools to neurophysiologists to steer cellular responses with unprecedented temporal and spatial resolution. The former takes advantage of light as an ultrashort trigger, whereas the latter is achieved by genetically encoding and directing photosensitive proteins to specific cell types. The most prominent among the optogenetics tools is channelrhodopsin (ChR), which was found to be the first light-gated ion channel of its kind (1, 2). This discovery paved the way for an exponentially growing number of neurophysiological applications, ranging from single cells to living animals (3). Light-gated ion permeation by ChR expands the various modes of action of the large family of microbial rhodopsins already comprising light-driven ion pumps and sensors (4). Among the various ChRs, which differ mostly in cation selectivity (3), ChR2 is used in the majority of optogenetic applications because of the higher expression yield in mammalian cells.A projection structure of the heptahelical ChR2 showed a dimer with the contact interface between helices C and D suggested to form the cation channel (5). More recently, a chimeric ChR (C1C2) was constructed by linking the last two helices (F and G) of ChR2 to the first five (A to E) of ChR1 and resolved by X-ray crystallography to 2.3 Å (6). The high-resolution structure confirmed the dimeric arrangement and identified an electronegative extracellular pore in each monomer framed by helices A, B, C, and G. Accompanying electrophysiological experiments on point mutants indicated r...

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The DC gate in Channelrhodopsin-2: crucial hydrogen bonding interaction between C128 and D156

Cited by 87 publications

References 18 publications

Gating mechanisms of a natural anion channelrhodopsin

Gating mechanisms of a natural anion channelrhodopsin

Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating

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