Based on the FTIR and UV-visible spectroscopic data, we deduced a six-state model of the ChR2 photocycle

Based on the FTIR and UV-visible spectroscopic data, we deduced a six-state model of the ChR2 photocycle. carboxylic part chain of Glu90is 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)3are light-gated cation channels (1,2) that share homology with additional microbial rhodopsins such as bacteriorhodopsin (BR), halorhodopsin (HR), and sensory rhodopsin (SR). In nature they serve as sensory photoreceptors for phobic reactions and phototaxis in green algae (3-5). The light-induced ion conductance prospects to depolarization of the cell membrane within milliseconds. Because of this house, 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 inCaenorhabditis elegans, Drosophila, zebrafish, chicken embryos, and mice (8-13). As is definitely standard 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 recombinantVolvoxchannelrhodopsin (VChR), purified from green monkey COS cells, comprises the two dark claims D470 and D480, characterized by a fine organized UV-visible absorption spectrum with maxima at 470 and 480 nm, respectively (14). These two states, which exist inside a pH-dependent equilibrium, are both converted by light via retinal isomerization and transient Schiff foundation deprotonation to the conducting state P510 or, under acidic conditions, to P530. These intermediates thermally unwind back to the dark state equilibrium inside a biphasic reaction on a millisecond time level. A similar model was deduced for ChR2 fromChlamydomonas reinhardtii(15), in which the conducting state, termed P520, was also observed to decay biexponentially with time constants of BFH772 10 ms and 5 s. No complete spectra and no early photocycle intermediates were analyzed with this study. Here we present more detailed spectroscopic studies on crazy typeChlamydomonasChR2 and the mutant E90Q, which is definitely 10 times more selective for Na+than BFH772 for H+as compared with the crazy type.4Because UV-visible studies solely provide information about the protonation state of the Schiff foundation and constraints of the retinal chromophore, we additionally applied Fourier transform infrared (FTIR) difference spectroscopy, which allowed us to analyze structural changes beyond the direct protein-chromophore connection. The FTIR difference spectra of formation and decay of three different intermediates are offered. Based on the FTIR and UV-visible spectroscopic data, we deduced a six-state model of the ChR2 photocycle. We provide evidence that molecular changes during this photocycle comprise retinal isomerization, Schiff foundation protonation change, changes in hydrogen bonding of the protonated carboxylic acid Glu90, and structural alterations of the protein backbone. As indicated from the spectra, these changes of the secondary structure are large compared with additional microbial rhodopsins but are similar with visual rhodopsin, which is a G-protein-coupled receptor. Such large changes are BFH772 ATN1 already observed in an intermediate cryotrapped at 80 K. FTIR difference spectroscopy indicated the glutamic acid part chain of Glu90changes its hydrogen bonding pattern during the photocycle. The late transitions, finally leading to the recovery of the dark state, comprise two intermediates with decay time constants of 6 and 40 s. In contrast to VChR, ChR2 does not display any pH-dependent equilibrium in the dark. == EXPERIMENTAL Methods == ChR2 Manifestation in COS-1 CellsFor manifestation in COS-1 cells (ATCC, CRL-1650), a human being codon-optimized synthetic ChopDNAfragment (related to amino acids 1-311 of the native protein; accession numberAF461397), plus the C-terminal ETSQVAPA sequence.