Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

We have isolated phytochrome B (phyB) and phyC mutants from rice (Oryza sativa) and have produced all combinations of double mutants. Seedlings of phyB and phyB phyC mutants exhibited a partial loss of sensitivity to continuous red light (Rc) but still showed significant deetiolation responses. The responses to Rc were completely canceled in phyA phyB double mutants. These results indicate that phyA and phyB act in a highly redundant manner to control deetiolation under Rc. Under continuous far-red light (FRc), phyA mutants showed partially impaired deetiolation, and phyA phyC double mutants showed no significant residual phytochrome responses, indicating that not only phyA but also phyC is involved in the photoperception of FRc in rice. Interestingly, the phyB phyC double mutant displayed clear R/FR reversibility in the pulse irradiation experiments, indicating that both phyA and phyB can mediate the low-fluence response for gene expression. Rice is a short-day plant, and we found that mutation in either phyB or phyC caused moderate early flowering under the long-day photoperiod, while monogenic phyA mutation had little effect on the flowering time. The phyA mutation, however, in combination with phyB or phyC mutation caused dramatic early flowering.

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These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.

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.

Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex. We find that synaphin promotes SNAREs to form precomplexes that oligomerize into higher order structures. A peptide from the central, syntaxin binding domain of synaphin competitively inhibits these two proteins from interacting and prevents SNARE complexes from oligomerizing. Injection of this peptide into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis. We propose that oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.

Channelrhodopsin-2 (ChR2), one of the archea-type rhodopsins from green algae, is a potentially useful optogenetic tool for restoring vision in patients with photoreceptor degeneration, such as retinitis pigmentosa. If the ChR2 gene is transferred to retinal ganglion cells (RGCs), which send visual information to the brain, the RGCs may be repurposed to act as photoreceptors. In this study, by using a transgenic rat expressing ChR2 specifically in the RGCs under the regulation of a Thy-1.2 promoter, we tested the possibility that direct photoactivation of RGCs could restore effective vision. Although the contrast sensitivities of the optomotor responses of transgenic rats were similar to those observed in the wild-type rats, they were enhanced for visual stimuli of low-spatial frequency after the degeneration of native photoreceptors. This result suggests that the visual signals derived from the ChR2-expressing RGCs were reinterpreted by the brain to form behavior-related vision.

Optogenetics is an optical technique that exploits visible light for selective neuromodulation with spatio-temporal precision. Despite enormous effort, the effective stimulation of targeted neurons, which are located in deeper structures of the nervous system, by visible light, remains a technical challenge. Compared to visible light, near-infrared illumination offers a higher depth of tissue penetration owing to a lower degree of light attenuation. Herein, an overview of advances in developing new modalities for neural circuitry modulation utilizing upconversion-nanoparticlemediated optogenetics is presented. These developments have led to minimally invasive optical stimulation and inhibition of neurons with substantially improved selectivity, sensitivity, and spatial resolution. The focus is to provide a comprehensive review of the mechanistic basis for evaluating upconversion parameters, which will be useful in designing, executing, and reporting optogenetic experiments.