[Show abstract][Hide abstract] ABSTRACT: The light absorption of a channelrhodopsin-2 (ChR2) is followed by conformational changes to the molecule, which allows the channel structure to become permeable to cations. Previously, a single point mutation in ChR2, which replaces glutamate residue 97 with a nonpolar alanine (E97A), was found to attenuate the photocurrent, suggesting that the E97 residue is involved in ion flux regulation. Here, the significance of E97 and its counterpart ChR1 (E136) were extensively studied by mutagenesis, whereby we replaced these glutamates with aspartate (D), glutamine (Q) or arginine (R). We found that the charge at this position strongly influences ion permeation and that the photocurrents were attenuated in the order of ChR2>E97D≈E97Q>E97R. We observed similar results with our chimeric/synthetic/artificial construct, ChR-wide receiver (ChRWR), which contains the first to fifth transmembrane helices of ChR1. The E-to-Q or E-to-R mutations, but not the E-to-D mutation, strongly retarded the sensitivity to the Gd(3+)-dependent blocking of the ChR1 or ChR2 channels. Our results suggest that the glutamate residue at this position lies in the outer pore, where it interacts with a cation to facilitate dehydration, and that this residue is the primary binding target of Gd(3+).
No preview · Article · Jun 2012 · Neuroscience Research
[Show abstract][Hide abstract] ABSTRACT: Channelrhodopsin-2 (ChR2), one of the algal light-gated cation channel rhodopsins, contains five peculiar glutamic acid residues in the N-terminal region corresponding to the second to third transmembrane helices. Here we made systematic mutations of these polar amino acid residues of ChR2 into nonpolar alanine, and evaluated their photocurrent properties. Amongst them, the photocurrent generated by the E97A mutation, ChR2(E97A), was much smaller than expected from its expression. The ChR2(E97A) photocurrent was similar to wild-type ChR2 in the kinetic profiles, the reversal potential and the dependency to the light power density. Our results suggest that the residue E97 is one of the molecular determinants involved in the ion flux regulation.
No preview · Article · Apr 2009 · Photochemical and Photobiological Sciences
[Show abstract][Hide abstract] ABSTRACT: A light signal is converted into an electrical one in a single molecule named channelrhodopsin, one of the archaea-type rhodopsins in unicellular green algae. Although highly homologous, two molecules of this family, channelrhodopsin-1 (ChR1) and -2 (ChR2), are distinct in photocurrent properties such as the wavelength sensitivity, desensitization, and turning-on and -off kinetics. However, the structures regulating these properties have not been completely identified. Photocurrents were analyzed for several chimera molecules made by replacing N-terminal segments of ChR2 with the homologous counterparts of ChR1. We found that the wavelength sensitivity of the photocurrent was red-shifted with negligible desensitization and slowed turning-on and -off kinetics when replacement was made with the segment containing the fifth transmembrane helix of ChR1. Therefore, this segment is involved in the determination of photocurrent properties, the wavelength sensitivity, and the kinetics characterizing ChR1 and ChR2. Eight amino acid residues differentiating this segment were exchanged one-by-one, and the photocurrent properties of each targeted mutant ChR2 were further analyzed. Among them, position Tyr(226)(ChR1)/Asn(187)(ChR2) is one of the molecular determinants involved in the wavelength sensitivity, desensitization, and turning-on and -off kinetics. It is suggested that these amino acid residues directly or indirectly interact with the chromophore as well as with the protein structure determining the photocurrent kinetics. Some of the chimera channelrhodopsins are suggested to have several advantages over the wild-type ChR2 in the introduction of light-induced membrane depolarization for the purpose of artificial stimulation of neurons in vivo and visual prosthesis for photoreceptor degeneration.
Preview · Article · Feb 2009 · Journal of Biological Chemistry