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...

Channelrhodopsin-2 (ChR2) is a member of the new class of light-gated ion channels which serve as phototaxis receptors in the green alga Chlamydomonas reinhardtii. The protein is employed in optogenetics where neural circuits are optically stimulated under high spatiotemporal control. Despite its rapidly growing use in physiological experiments, the reaction mechanism of ChR2 is poorly understood. Here, we applied vibrational spectroscopy to trace structural changes of ChR2 after light-excitation of the retinal chromophore. FT-IR difference spectra of the various photocycle intermediates revealed that stages of the photoreaction preceding (P(1) state) and succeeding (P(4)) the conductive state of the channel (P(3)) are associated with large conformational changes of the protein backbone as indicate by strong differences in the amide I bands. Critical hydrogen-bonding changes of protonated carboxylic amino acid side chains (D156, E90) were detected and discussed with regard to the functional mechanism. We used the C128T mutant where the lifetime of P(3) is prolonged and applied FT-IR and resonance Raman spectroscopy to study the conductive P(3) state of ChR2. Finally, a mechanistic model is proposed that links the observed structural changes of ChR2 to the changes in the channel's conductance.

Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule.