Driving forces of proton-pumping rhodopsins.

Proton-pumping rhodopsins are light-driven proton transporters that have been discovered from various microbiota. They are categorized into two groups: outward-directed and inward-directed proton pumps. Although the directions of transport are opposite, they are active proton transporters that create an H gradient across a membrane.

The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization.

[How to Choose the Best Optogenetic Tool for Your Research].

All-optical methods that provide deeper understanding of neural activity are currently being developed. Optogenetics is a biological technique useful to control neuronal activity or life phenomena using light. Microbial rhodopsins are light-activated membrane proteins used as optogenetic tools.

Optogenetic manipulation of neuronal and cardiomyocyte functions in zebrafish using microbial rhodopsins and adenylyl cyclases.

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control cell and tissue functions in vivo. We exploited CCR4 and ChR, cation channelrhodopsins from algae, GC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (PAC) or bacteria (PAC), to control cell functions in zebrafish. Optical activation of CCR4 and ChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, induced swimming behavior at relatively short latencies, whereas activation of GC1 or PACs achieved it at long latencies.

Light-Driven Chloride and Sulfate Pump with Two Different Transport Modes.

Ion pumps are membrane proteins that actively translocate ions by using energy. All known pumps bind ions in the resting state, and external energy allows ion transport through protein structural changes. The light-driven sodium-ion pump rhodopsin 2 (KR2) is an exceptional case in which ion binding follows the energy input.

A light-gated cation channel with high reactivity to weak light.

The cryptophyte algae, Guillardia theta, possesses 46 genes that are homologous to microbial rhodopsins. Five of them are functionally light-gated cation channelrhodopsins (GtCCR1-5) that are phylogenetically distinct from chlorophyte channelrhodopsins (ChRs) such as ChR2 from Chlamydomonas reinhardtii. In this study, we report the ion channel properties of these five CCRs and compared them with ChR2 and other ChRs widely used in optogenetics.

Functional assay of light-induced ion-transport by rhodopsins.

Microbial rhodopsins are photoreceptive membrane proteins found from diverse microorganisms such as archaea, eubacteria, eukaryotes and viruses. Many microbial rhodopsins possess ion-transport activity by light, such as channels and pumps, and ion-transporting rhodopsins are important tools in optogenetics that control animal behavior by light. Historically, molecular mechanism of rhodopsins has been studied by spectroscopic methods for purified proteins.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter.

Ion transport activity and optogenetics capability of light-driven Na+-pump KR2.

KR2 from marine bacteria Krokinobacter eikastus is a light-driven Na+ pumping rhodopsin family (NaRs) member that actively transports Na+ and/or H+ depending on the ionic state. We here report electrophysiological studies on KR2 to address ion-transport properties under various electrochemical potentials of Δ[Na+], ΔpH, membrane voltage and light quality, because the contributions of these on the pumping activity were less understood so far. After transient expression of KR2 in mammalian cultured cells (ND7/23 cells), photocurrents were measured by whole-cell patch clamp under various intracellular Na+ and pH conditions.

Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens.

Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties.

Gate-keeper of ion transport-a highly conserved helix-3 tryptophan in a channelrhodopsin chimera, C1C2/ChRWR.

Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all- retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown.

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H pumps.

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H pump.

Novel optogenetics tool: Gt_CCR4, a light-gated cation channel with high reactivity to weak light.

Optogenetics is a growing technique which allows manipulation of biological events simply by illumination. The technique is appreciated especially in the neuroscience field because of its availability in controlling neuronal functions. A light-gated cation channel, Cr_ChR2 from Chlamydomonas reinhardtii, is the first and mostly applied to optogenetics for activating neuronal excitability.

Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics. They are widely present in bacteria, archaea, algae and algal viruses. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins.

The regulatory mechanism of ion permeation through a channelrhodopsin derived from Mesostigma viride (MvChR1).

The five glutamate (E) residues of transmembrane (TM)-2 of channelrhodopsin (CrChR)-2 are conserved among several members of the ChR family. A point mutation of one of them, E97, to a nonpolar alanine (E97A) reduced the photocurrent amplitude without influencing other photocurrent properties. The charge at this position is also the determinant of the Gd(3+)-dependent block of the channel.

Near-infrared (NIR) up-conversion optogenetics.

Non-invasive remote control technologies designed to manipulate neural functions have been long-awaited for the comprehensive and quantitative understanding of neuronal network in the brain as well as for the therapy of neurological disorders. Recently, it has become possible for the neuronal activity to be optically manipulated using biological photo-reactive molecules such as channelrhodopsin (ChR)-2. However, ChR2 and its relatives are mostly reactive to visible light, which does not effectively penetrate through biological tissues.

Structural basis for Na(+) transport mechanism by a light-driven Na(+) pump.

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.

Kinetic evaluation of photosensitivity in bi-stable variants of chimeric channelrhodopsins.

Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A).

Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion.

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca(2+) elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse.