Kinetics of proton release and uptake by channelrhodopsin‐2

Nack, Melanie; Radu, Ionela; Schultz, Bernd-Joachim; Resler, Tom; Schlesinger, Ramona; Bondar, Ana‐Nicoleta; Val, Coral del; Abbruzzetti, Stefania; Viappiani, Cristiano; Bamann, Christian; Bamberg, Ernst; Heberle, Joachim

doi:10.1016/j.febslet.2012.03.047

Cited by 29 publications

(42 citation statements)

References 49 publications

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“…The latter two groups are 9.7 Å apart, which is comparable to the 12.0-Å distance between the SB and D96 in bR (46). We recently monitored transient pH changes in the aqueous medium with the help of a pH-indicating dye (47). Proton release to the bulk aqueous medium was detected with the rise of the P 3 520 state.…”

Section: Discussionmentioning

confidence: 96%

“…The resulting proteindetergent micelle sufficiently mimics the properties of the lipid bilayer, as was concluded in previous spectroscopic studies (18,51). For experiments measuring the transient pH changes of E90A, the buffer was substituted with 15 μM bromoxylenol blue, a pH indicator (47). Samples used for FTIR spectroscopy were concentrated to ∼4 mg/mL ChR2 in an aqueous solution of 5 mM NaCl and 5 mM Hepes at pH 7.4, and ∼8 μL was dried on top of a BaF 2 window.…”

Section: Methodsmentioning

confidence: 99%

“…The photoreaction in the IR and UV/Vis experiments was induced by a short laser pulse emitted by a neodymium yttrium aluminum garnet laser (Nd:YAG from Quanta-Ray; Spectra-Physics), which drives an optical parametric oscillator (OPTA). The resulting emission was set to a wavelength of 450 nm with a 10-ns pulse width and a 3-mJ/cm 2 energy density at the sample, leading to a photoconversion of about 10% of the molecules (47). Time-resolved step-scan FTIR difference spectra were recorded in the time range of 6.25 μs to 125 ms. Rapid-scan FTIR spectroscopy was used to trace the slower kinetic range (90 ms to 95 s).…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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

414

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“…ChR belongs, as bR, to the family of microbial rhodopsins but in contrast to bR, much less is known about its functional mechanism 52 . ChR2 combines its function as an ion channel with proton-pumping activity 46,47 . Recently, we applied time-resolved step-scan FT-IR spectroscopy to resolve intraprotein proton transfer reactions and the dynamics of protein backbone conformation changes in ChR2 48 .…”

Section: Introductionmentioning

confidence: 99%

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Lórenz-Fonfrı́a

Heberle

2014

JoVE

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Citation: Lórenz-Fonfría, V.A., Heberle, J. Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Stepscan Fourier-transform Infrared Spectroscopy. J. Vis. Exp. (88), e51622, doi:10.3791/51622 (2014). AbstractMonitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~10 2 -10 3 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10 -4 , sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/ or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

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“…Schiff base deprotonation leads to the M-like state P 390 2 (10,11). This state is followed by the redshifted intermediate P state (τ = 24 s), which is referred to as the desensitized state with a spectral characteristic similar to the ground state (11,42). In addition, time-resolved FTIR spectroscopy indicated that P 520 3 could partially convert directly to the ground state (14).…”

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confidence: 99%

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Becker‐Baldus

Bamann

Saxena

et al. 2015

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

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Channelrhodopsin-2 from Chlamydomonas reinhardtii is a lightgated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15 N-labeled channelrhodopsin-2 carrying 14,15-13 C 2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P 500 1 K-like state, the slowly decaying late intermediate P 480 4 , and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.ince their discovery (1), channelrhodopsins (ChRs) have generated enormous interest because of the rapid development of their applications in optogenetics (2-7). Commonly, ChR2 from Chlamydomonas reinhardtii (8) and its variants are used thanks to their favorable expression levels. They are the only proteins known today functioning as light-gated ion channels (Fig. 1A). Like other microbial retinal proteins, they undergo a periodic photocycle. In ChRs, this photocycle is coupled to channel opening and closing as revealed in electrophysiological recordings (8). A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species terme...

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Kinetics of proton release and uptake by channelrhodopsin‐2

Cited by 29 publications

References 49 publications

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

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

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

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