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.
Highlights
Light is perceived by many living organisms on the earth as vital information
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
A possible molecular determinant was identified in the fifth transmembrane helix, which is involved in both the light absorbance and the photocurrent kinetics
Summary
Light is perceived by many living organisms on the earth as vital information. In the case of vertebrates, including human beings, the rhodopsins are the molecules involved in the light perception of the photoreceptor cells in the retina (1–3). Each rhodopsin is a seven-pass transmembrane molecule homologous to G-protein-linked receptors and activated by a photoisomerization of a covalently attached chromophore, 11-cis-retinal, to all-trans configuration. Two rhodopsins named channelrhodopsin-1 (ChR1) and -2 (ChR2) were identified in a green alga Chlamydomonas reinhardtii and extensively studied (7, 8, 10, 11). We replaced the N-terminal segments of ChR2 with the homologous counterparts of ChR1 and generated several chimeras. These chimeras generated photocurrents showing intermediate properties between ChR1 and ChR2. A possible molecular determinant was identified in the fifth transmembrane helix, which is involved in both the light absorbance and the photocurrent kinetics. Determinants Differentiating Channelrhodopsin Photocurrents be more optimal than ChR2 to depolarize exogenously expressed cells by light
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