Search

Even though microbial photosensitive proteins have been used for optogenetics, their use should be optimized to precisely control second messengersin vivo. We exploitedGtCCR4andKnChR, cation channelrhodopsins from algae,BeGC1, a guanylyl cyclase rhodopsin from a fungus, and photoactivated adenylyl cyclases (PACs) from cyanobacteria (OαPAC) or bacteria (bPAC), to control cell functions in zebrafish. Optical activation ofGtCCR4 andKnChR in the hindbrain reticulospinal V2a neurons, which are involved in locomotion, immediately induced swimming behavior, whereas activation ofBeGC1 or PACs was achieved at a short latency.KnChR had the highest locomotioninducing activity of all the channelrhodopsins examined. Activation ofGtCCR4 andKnChR in cardiomyocytes induced cardiac arrest, whereas activation ofbPAC gradually induced bradycardia.KnChR activation led to an increase in intracellular Ca2+in the heart, suggesting that depolarization caused cardiac arrest. These data suggest that these optogenetic tools can be used to reveal the roles of second messengers in various cell types in vertebrates.Impact statementWe identified efficient and useful microbial channelrhodopsin, guanylyl cyclase rhodopsin, and photoactivated adenylyl cyclase that regulate neural activity and cardiac function in zebrafish.Major subject areasNeuroscience, Cell biologyResearch organismZebrafish (Danio rerio)

Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all-trans 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. Here, we replaced the W163 (BR W86 counterpart) of a channelrhodopsin (ChR), C1C2/ChRWR, which is a chimera between ChR1 and 2, with a smaller aromatic residue, Phe to verify its role in the ion transport. Under whole-cell patch clamp recordings from the ND7/23 cells that were transfected with the DNA plasmid coding human codon optimized C1C2/ChRWR (hWR) or its W163F mutant (hWR-W163F), the photocurrents were evoked by a pulsatile light at 475 nm. The ion-transporting activity of hWR was strongly altered by the W163F mutation in 3 points: (1) the H+ leak at positive membrane potential (V m) and its light-adaptation, (2) the attenuation of cation channel activity and (3) the manifestation of outward H+ pump activity. All of these results strongly suggest that W163 has a role in stabilizing the structure involved in the gating-on and -off of the cation channel, the role of "gate keeper". We can attribute the attenuation of cation channel activity to the incomplete gating-on and the H+ leak to the incomplete gating-off.

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. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required., Tashiro et al. describe a new channelrhodopsin variant from a terrestrial algal species and the role of the C-terminal domain in regulatory function. This far-blue-shifted channelrhodopsin may contribute to optogenetic tool research in the future.

Channelrhodopsins are a family of microbial rhodopsins that function as a light-gated ion channel. We report the molecular properties of a novel channelrhodopsin KnRh3 from an evolutionary important filamentous terrestrial alga Klebsormidium nitens. KnRh3 is constituted of a 7-transmembrane domain, followed by a long C-terminus moiety that encodes a peptidoglycan binding domain (FimV). When functionally expressed in mammalian cells, KnRh3 showed light-induced cation channel currents. The maximum action spectrum exhibited was at 430 nm and 460 nm, the former making KnRh3 one of the most blue-shifted channelrhodopsins characterized thus far. The channel closure rate was relatively fast (τ0ff = 10 ms). Surprisingly, photocurrent kinetics were affected by the C-terminus moiety of KnRh3. When this moiety was truncated to various lengths, this prolonged the channel open lifetime by more than 10-fold. We identified two arginine residues, R287 and R291, those are crucial for altering the kinetics. We propose that electrostatic interaction between the 7-TM domain and the C-terminus domain accelerates the photocycle. The most blue-shifted action spectrum of KnRh3 serves as a novel prototype of channelrhodopsin for studying the molecular mechanism of color tuning. In addition, KnRh3 would expand the optogenetics tool kit, especially for when short wavelength excitation is required.

Schizorhodopsins, recently discovered rhodopsins in Asgard archaea, 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 Escherichia coli 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. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

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. In addition, the molecular mechanism of Cr_ChR2 has been intensively studied by electrophysiology, spectroscopy, X-ray structural studies, etc. Novel cation channelrhodopsins from Guillardia theta, namely, Gt_CCR1–4, were discovered in 2016 and 2017. These channelrhodopsins are more homologous to haloarchaeal rhodopsins, particularly the proton pumps. Thus these cryptophyte-type light-gated cation channels are structurally and mechanistically distinct from chlorophyte channelrhodopsin such as Cr_ChR2. We here compared the photocurrent properties, cation selectivity, and kinetics between well-known Cr_ChR2 and Gt_CCR4. The light sensitivity of Gt_CCR4 is significantly higher than that of Cr_ChR2, while the channel open lifetime is in the same range as that of Cr_ChR2. Gt_CCR4 shows high Na(+) selectivity in which the selectivity ratio for Na(+) was 37-fold larger than that for Cr_ChR2, which primarily conducts H(+). On the other hand, Gt_CCR4 conducted almost no H(+) and no Ca(2+) under physiological conditions. Other unique features and the applicability of Gt_CCR4 for optogenetics were discussed.

We previously reported a cation channelrhodopsin, Gt_CCR4, which is one of the 44 types of microbial rhodopsins from a cryptophyte flagellate, Guillardia theta. Due to the modest homology of amino acid sequences with a chlorophyte channelrhodopsin such as Cr_ChR2 from Chlamydomonas reinhardtii, it has been proposed that a family of cryptophyte channelrhodopsin, including Gt_CCR4, has a distinct molecular mechanism for channel gating and ion permeation. In this study, we compared the photocurrent properties, cation selectivity and kinetics between well-known Cr_ChR2 and Gt_CCR4 by a conventional path clamp method. Large and stable light-induced cation conduction by Gt_CCR4 at the maximum absorbing wavelength (530 nm) was observed with only small inactivation (15%), whereas the photocurrent of Cr_ChR2 exhibited significant inactivation (50%) and desensitization. The light sensitivity of Gt_CCR4 was higher (EC50 = 0.13 mW/mm2) than that of Cr_ChR2 (EC50 = 0.80 mW/mm2) while the channel open life time (photocycle speed) was in the same range as that of Cr_ChR2 (25~30 ms for Gt_CCR4 and 10~15 ms for Cr_ChR2). This observation implies that Gt_CCR4 enables optical neuronal spiking with weak light in high temporal resolution when applied in neuroscience. Furthermore, we demonstrated high Na+ selectivity of Gt_CCR4 in which the selectivity ratio for Na+ was 37-fold larger than that for Cr_ChR2, which primarily conducts H+. On the other hand, Gt_CCR4 conducted almost no H+ and no Ca2+ under physiological conditions. These results suggest that ion selectivity in Gt_CCR4 is distinct from that in Cr_ChR2. In addition, a unique red-absorbing and stable intermediate in the photocycle was observed, indicating a photochromic property of Gt_CCR4.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics

To determine whether fishes respond to menthol, Japanese medaka Oryzias latipes, goldfish Carassius auratus, and zebrafish Danio rerio were exposed to various types of menthol receptors agonists and the behavioral responses to these drugs were observed. Waterbone application of dl-menthol (0.5 mM) induced surgical anesthesia in 100% of medaka, 90% of goldfish, and 100% of zebrafish. The percentage of response increased dose-dependently from 0.2 mM to 0.5 mM. There were no differences in either percentage or latency of the response in surgical anesthesia among dl-, d-, and l- types of menthol. A high (3.0 mM) concentration of any of the three types of menthol induced rapid movement fol- lowed by the anesthetic response. Rapid movement was observed with allyl isothiocyanate, a cold nociceptor agonist, but not with icilin, a cold receptor agonist, in medaka and goldfish. Both allyl isothiocyanate and icilin failed to induce surgi- cal anesthesia. To determine the involvement of � -aminobutyric acid (GABA) system in menthol-induced surgical anes- thesia, the effect of the receptors antagonist for the GABAA was tested. Pretreatment with a specific GABAA receptor an- tagonist prolonged the latency of the anesthetic response to menthol, but not to cold-water stimulation, in medaka and goldfish. These results demonstrate that menthol can play a role in the induction of surgical anesthesia in fishes, related at least in part to the activation of GABAA receptors, and of rapid movement possibly via cold nociceptors.

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. It has thus been suggested that E97 interacts with hydrated cations to facilitate their permeation and that these residues are the primary binding sites of Gd(3+). However, the counterpart of this position is alanine for MvChR1 from Mesostigma viride. Here we investigated the ion permeation and the Gd(3+)-dependent channel block of MvChR1. We found that the high-affinity binding site of Gd(3+) was absent in MvChR1, but was dependent on the negativity at this position. However, the ion permeation through the channel was markedly interfered with a negative charge at this position. Based on these findings, it is proposed that the ions can pass through the pore with minimal interaction with this position.

Non-invasive remote control technologies designed to manipulate neural functions for a comprehensive and quantitative understanding of the neuronal network in the brain as well as for the therapy of neurological disorders have long been awaited. Recently, it has become possible to optically manipulate the neuronal activity using biological photo-reactive molecules such as channelrhodopsin-2 (ChR2). However, ChR2 and its relatives are mostly reactive to visible light which does not effectively penetrate through biological tissues. In contrast, near-infrared (NIR) light penetrates deep into the tissues because biological systems are almost transparent to light within this so-called ‘imaging window’. Here we used lanthanide nanoparticles (LNPs), which are composed of rare-earth elements, as luminous bodies to activate channelrhodopsins (ChRs) since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Neuron-glioma-hybrid ND-7/23 cells were cultured with LNP(NaYF4:Sc/Yb/Er) particles (peak emission, 543 nm) and transfected to express C1V1 (peak absorbance, 539 nm), a chimera of ChR1 and VChR1. The photocurrents were generated in response to NIR laser light (976 nm) to a level comparable to that evoked by a filtered Hg lamp (530-550 nm). NIR light pulses also evoked action potentials in the cultured neurons that expressed C1V1. It is suggested that the green luminescent light emitted from LNPs effectively activated C1V1 to generate the photocurrent. With the optimization of LNPs, acceptor photo-reactive biomolecules and optics, this system could be applied to non-invasively actuate neurons deep in the brain.

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). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0-0.12 mW mm(-2) of blue LED light or to 0-0.33 mW mm(-2) of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τm > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.

Recent optical neuro-research methods have several advantages over conventional techniques: high resolution in space and time, together with direct measurement and manipulation of cell functions by light. The use of fluorescent proteins, bioluminescence systems, and light-sensitive proteins facilitates the optical methods in combination with genetic engineering techniques. Techniques involving the application of light-sensitive proteins are collectively termed ‘optogenetics’ because they combine optics and genetics. Light-sensitive proteins, either natural or synthetic, are termed ‘optogenetic molecular reagents’ (OMRs) or ‘optogenetic molecular tools’ if they are used as tools for optogenetics. These techniques are receiving recognition as enabling breakthroughs in neuroscience research. In this chapter, the topics and the prospects of optogenetics are discussed from three view points: the optimization of OMRs, the optimization of gene-targeting methods, and the optimization of optics.

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. Known microbial rhodopsins were classified into two groups, either outward proton pumps or inward chloride pumps, until the recent discovery of a light-driven Na+-pumping rhodopsin from the marine bacterium Krokinobacter eikastus. This novel protein, termed KR2, is attracting attention as a potential tool for use in optogenetics: its activation would change the sodium concentration of a targeted cell, not just the pH or chloride concentration. Now Osamu Nureki and colleagues have solved two X-ray crystal structures of KR2 and they use them to propose a working model for Na+ transport. Based on these structures the authors have designed several mutants of KR2 and successfully engineered a K+-transporting pump.

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. In contrast, near-infrared (NIR) light (650–1450 nm) penetrates deep into the tissues because biological systems are almost transparent to light within this so-called ‘imaging window’. Here we used lanthanide nanoparticles (LNPs), composed of rare-earth elements, as luminous bodies to activate ChRs since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Here, we created a new type of optogenetic system which consists of the donor LNPs and the acceptor ChRs. The NIR laser irradiation emitted visible light from LNPs, then induced the photo-reactive responses in the near-by cells that expressed ChRs. However, there remains room for large improvements in the energy efficiency of the LNP-ChR system.