Monitoring Light-induced Structural Changes of Channelrhodopsin-2 by UV-visible and Fourier Transform Infrared Spectroscopy

Abstract

Channelrhodopsin-2 (ChR2) is a microbial type rhodopsin and a light-gated cation channel that controls phototaxis in Chlamydomonas. We expressed ChR2 in COS-cells, purified it, and subsequently investigated this unusual photoreceptor by flash photolysis and UV-visible and Fourier transform infrared difference spectroscopy. Several transient photoproducts of the wild type ChR2 were identified, and their kinetics and molecular properties were compared with those of the ChR2 mutant E90Q. Based on the spectroscopic data we developed a model of the photocycle comprising six distinguishable intermediates. This photocycle shows similarities to the photocycle of the ChR2-related Channelrhodopsin of Volvox but also displays significant differences. We show that molecular changes include retinal isomerization, changes in hydrogen bonding of carboxylic acids, and large alterations of the protein backbone structure. These alterations are stronger than those observed in the photocycle of other microbial rhodopsins like bacteriorhodopsin and are related to those occurring in animal rhodopsins. UV-visible and Fourier transform infrared difference spectroscopy revealed two late intermediates with different time constants of ‫؍‬ 6 and 40 s that exist during the recovery of the dark state. The carboxylic side chain of Glu 90 is involved in the slow transition. The molecular changes during the ChR2 photocycle are discussed with respect to other members of the rhodopsin family. Channelrhodopsins (ChRs)3 are light-gated cation channels (1, 2) that share homology with other microbial rhodopsins such as bacteriorhodopsin (BR), halorhodopsin (HR), and sensory rhodopsin (SR). In nature they serve as sensory photoreceptors for phobic responses and phototaxis in green algae (3-5). The light-induced ion conductance leads to depolarization of the cell membrane within milliseconds. Because of this property, in recent years ChRs have been widely used in the neuroscience field as a tool for depolarization of selected cell types or cell ensembles (6, 7). Furthermore, channelrhodopsins were used to control neuronal activity in Caenorhabditis elegans, Drosophila, zebrafish, chicken embryos, and mice (8 -13).As is typical for rhodopsins, light absorption induces isomerization of the ChR-chromophore with subsequent conformational changes in the protein (photocycle). Based on UV-visible spectroscopic and electrophysiological measurements, several schemes for this photocycle have been presented. A recent model for recombinant Volvox channelrhodopsin (VChR), purified from green monkey COS cells, comprises the two dark states D470 and D480, characterized by a fine structured UVvisible absorption spectrum with maxima at 470 and 480 nm, respectively (14). These two states, which exist in a pH-dependent equilibrium, are both converted by light via retinal isomerization and transient Schiff base deprotonation to the conducting state P510 or, under acidic conditions, to P530. These intermediates thermally relax back to the dark state equilibrium in a biphas...

Astrocytes Display Complex and Localized Calcium Responses to Single-Neuron Stimulation in the Hippocampus

“…However, under these conditions, the frequency of AP spiking behavior was rapidly attenuated within a few seconds, suggesting neuronal fatigue or ChR2 dark state (Ritter et al, 2008). In contrast, 568 nm Kr/Ar laser light did not trigger APs (Fig.…”

Photochemical reaction cycle transitions during anion channelrhodopsin gating

“…The correspondence between the spectral intermediates and channel states in ACRs and CCRs appears to be quite different, which further confirms that these proteins represent two distinct families of microbial rhodopsins. In CCRs (or at least in the best studied CrChR2), deprotonation of the Schiff base occurs much earlier than channel opening, and, therefore, the conducting state of the channel was initially linked to the next red-shifted intermediate of the photocycle (P520) (20,21). At least in the slow C128X mutants, photocurrents are quenched not only by green, but also by violet light (absorbed by an M-like intermediate, P-390), which led to the conclusion that CrChR2 opens already in the M state (6).…”

Polarizable Embedding Density Matrix Renormalization Group

“…The experimental gas-phase peak maximum is between 2.00 eV and 2.03 eV [141,142], whereas it is shifted to 2.58 and 2.70 eV in the protein [143,144]. Only srDFT captures the neglected dynamical correlation in such a way that for DMRG-srPBE and PE-DMRG-srPBE we finally obtained improved excitation energies of 2.75 eV in vacuum and 3.27 eV in the protein, respectively.…”