Molecular Determinants Differentiating Photocurrent Properties of Two Channelrhodopsins from Chlamydomonas

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 oneby-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.

Visually induced initiation of Drosophila innate courtship-like following pursuit is mediated by central excitatory state

“…We reasoned that the chemosensory stimuli offered to the male by tapping increased the excitability of certain neurons critical for initiating courtship, so that subsequent visual input could induce a sustained courtship-like pursuit. We therefore attempted to replace the chemosensory stimuli for courtship induction with optogenetic stimulation of neurons via channelrhodopsin-wide receiver (ChRWR), which can be activated by blue light irradiation 10 . Indeed, courtship-like pursuit was induced in a male without tapping when dsx-expressing neurons were stimulated via ChRWR while a moving light spot was being presented in front of him; the male ran to follow the target and extended/vibrated a wing to sing, while gradually turning his body axis (shown as Dx) according to the target position ( Fig.…”

“…*Po0.05, ***Po0.001, Mann-Whitney's U-test. 10 . The DNA fragment containing ChRWR::Venus was excised with the EcoR I and Not I restriction enzymes, then introduced to EcoR I-Not I restriction sites of the pUAST vector.…”

Selective Manipulation of Neural Circuits

“…Through molecular engineering, these tools have been optimized to allow for faster alteration of channels [114,115], response to different wavelengths [116][117][118][119][120], more robust gene expression [114,117,121,122], and channels with stepwise kinetics [117,123,124]. Having tools activated with different wavelengths makes it possible to manipulate the same neuron bidirectionally with both excitatory and inhibitory channels.…”

Optogenetics in neuroscience: what we gain from studies in mammals

“…However, these proteins are inefficient in terms of stable expression and the control of spiking properties and photocurrent. In order to optimize these optogenetic tools, many modified strategies have been offered, including genetic variants [10] , chimeras [11] , and structural engineering [12] . Recently, researchers have developed new optogenetic tools to regulate cellular biochemical signaling [13] .…”