Elsevier BV Acta Medica Okayama 0021-9258 302 8 2026 Proton pump rhodopsins for optogenetic manipulation of biological activities and beyond 113318 EN Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Miyu Inokuchi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Water (H2O), the principal component of living organisms including humans, dissociates into H+ and OH- in aqueous environments, and the resulting H+ concentration determines both cellular pH and the proton motive force (PMF) across cellular membranes. These physicochemical parameters are fundamental regulators of a wide range of biological processes. Optogenetics enables the manipulation of biological and cellular functions using light, typically through the ectopic expression of microbial rhodopsins as photoreceptive proteins in target cells or organs. This review provides a comprehensive overview of optogenetic studies employing H+ pump rhodopsins in diverse biological systems and highlights their growing relevance to neuroscience, cell biology, bioengineering, and therapeutic research. Notably, optical control of H+ concentration allows the precise modulation of neural and non-neural activities in animals and bacteria, highlighting the broad applicability and significant potential of H+ pump rhodopsins for optogenetics. Based on previous and current studies, we discuss how further expansion of the molecular toolkit of H+ pump rhodopsins could enable increasingly fine-tuned manipulation of intracellular pH dynamics and PMF for probing cellular physiology and designing next-generation therapeutic strategies and consider emerging directions that extend beyond classical optogenetics toward the optical control of bioenergetic and chemical processes. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 1948-7185 16 23 2025 Ultrafast Protein Dynamics Prior to Retinal Photoisomerization in Microbial Rhodopsins 5732 5738 EN Shinya Tahara Molecular Spectroscopy Laboratory, RIKEN Rika Kurihara Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hikaru Kuramochi Molecular Spectroscopy Laboratory, RIKEN Satoshi Takeuchi Molecular Spectroscopy Laboratory, RIKEN Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tahei Tahara Molecular Spectroscopy Laboratory, RIKEN Rhodopsins, an important group of photoreceptor proteins, possess a retinal chromophore. It has long been believed that the photoisomerization of the chromophore is the initial event triggering its photocycle. However, we recently reported that protein structural changes around the chromophore precede photoisomerization in bacteriorhodopsin (BR). In this study, we performed deep-ultraviolet femtosecond stimulated Raman measurements for H+-pump rhodopsin (RxR) and photosensor rhodopsin (NpSRII) to investigate whether these ultrafast protein dynamics are common among rhodopsins. We observed that the protein structural changes occur within 0.2 ps after photoexcitation and precede the chromophore photoisomerization, similar to the case for BR. This strongly indicates that the protein structural change precedes the photoisomerization in rhodopsins, regardless of the difference in their origins and functions. Furthermore, we found that only limited protein changes occur on the time scale of the photoisomerization, suggesting that the protein environment is already optimized for the structural change of the chromophore. These observations raise the possibility that the ultrafast protein change arranges the environment around the chromophore to facilitate the photoisomerization. No potential conflict of interest relevant to this article was reported.

Biophysical Society of Japan Acta Medica Okayama 2189-4779 22 3 2025 Origin of the unique topology of the triangular water cluster in <i>Rubrobacter xylanophilus</i> rhodopsin e220018 EN Tomoyasu Noji Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo Masaki Tsujimura Department of Advanced Interdisciplinary Studies, The University of Tokyo Keisuke Saito Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroshi Ishikita Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo The crystal structure of Rubrobacter xylanophilus rhodopsin (RxR) reveals a triangular cluster of three water molecules (W413, W415, and W419) at the extracellular proton-release site, near Glu187 and Glu197. Using a quantum mechanical/molecular mechanical approach, we identified the structural nature of this unique water cluster. The triangular shape is best reproduced when all three water molecules are neutral H2O with protonated Glu187 and deprotonated Glu197. Attempts to place H3O+ at any of these water molecules result in spontaneous proton transfer to one of the acidic residues and significant distortion from the crystal structure. The plane defined by the triangular water cluster extends into the guanidinium plane of Arg71, with both aligned along the W413...W419 axis. This extended plane lies nearly perpendicular to a five-membered, ring-like H-bond network involving two carboxyl oxygen atoms from Glu187 and one from Glu197. The resulting bipartite planar architecture, defined by the water triangle, Arg71, and the Glu187/Glu197 network may reflect the exceptional thermal stability in RxR. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 0002-7863 2025 Optogenetic Cancer Therapy Using the Light-Driven Outward Proton Pump Rhodopsin Archaerhodopsin-3 (AR3) EN Shin Nakao Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keita Sato Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naoya Kemmotsu Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hideyo Ohuchi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Togashi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Medicines used for cancer treatment often cause serious side effects by damaging normal cells due to nonspecific diffusion. To address this issue, we previously developed an optical method to induce apoptotic cell death via intracellular pH alkalinization using the outward proton pump rhodopsin, Archaerhodopsin-3 (AR3) in various noncancer model cells in vitro and in vivo. In this study, we applied this method to cancer cells and tumors to evaluate its potential as an anticancer therapeutic strategy. First, we confirmed that AR3-expressing murine cancer cell lines (MC38, B16F10) showed apoptotic cell death upon green light irradiation, as indicated by increased levels of cell death and apoptosis-related markers. Next, we established stable AR3-expressing MC38 and B16F10 cells by using viral vectors. When these AR3-expressing cells were subcutaneously transplanted into C57BL/6 mice, the resulting tumors initially grew at a rate comparable to that of control tumors lacking AR3 expression or light stimulation. However, upon green light irradiation, AR3-expressing tumors exhibited either a marked reduction in size or significantly suppressed growth, accompanied by the induction of apoptosis signals and decreased proliferation signals. These results demonstrate that AR3-mediated cell death has potent antitumor effects both in vitro and in vivo. This optical method thus holds promise as a novel cancer therapy with potentially reduced side effects. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 1751-7362 18 1 2024 Cyanorhodopsin-II represents a yellow-absorbing proton-pumping rhodopsin clade within cyanobacteria wrae175 EN Masumi Hasegawa-Takano Atmosphere and Ocean Research Institute, The University of Tokyo Toshiaki Hosaka Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Nishimura Atmosphere and Ocean Research Institute, The University of Tokyo Marie Kurihara Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yu Nakajima Atmosphere and Ocean Research Institute, The University of Tokyo Yoshiko Ishizuka-Katsura Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research Tomomi Kimura-Someya Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research Mikako Shirouzu Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Susumu Yoshizawa Atmosphere and Ocean Research Institute, The University of Tokyo Microbial rhodopsins are prevalent in many cyanobacterial groups as a light-energy-harvesting system in addition to the photosynthetic system. It has been suggested that this dual system allows efficient capture of sunlight energy using complementary ranges of absorption wavelengths. However, the diversity of cyanobacterial rhodopsins, particularly in accumulated metagenomic data, remains underexplored. Here, we used a metagenomic mining approach, which led to the identification of a novel rhodopsin clade unique to cyanobacteria, cyanorhodopsin-II (CyR-II). CyR-IIs function as light-driven outward H+ pumps. CyR-IIs, together with previously identified cyanorhodopsins (CyRs) and cyanobacterial halorhodopsins (CyHRs), constitute cyanobacterial ion-pumping rhodopsins (CyipRs), a phylogenetically distinct family of rhodopsins. The CyR-II clade is further divided into two subclades, YCyR-II and GCyR-II, based on their specific absorption wavelength. YCyR-II absorbed yellow light (λmax = 570 nm), whereas GCyR-II absorbed green light (λmax = 550 nm). X-ray crystallography and mutational analysis revealed that the difference in absorption wavelengths is attributable to slight changes in the side chain structure near the retinal chromophore. The evolutionary trajectory of cyanobacterial rhodopsins suggests that the function and light-absorbing range of these rhodopsins have been adapted to a wide range of habitats with variable light and environmental conditions. Collectively, these findings shed light on the importance of rhodopsins in the evolution and environmental adaptation of cyanobacteria. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0021-9258 301 7 2025 A repertoire of visible light–sensitive opsins in the deep-sea hydrothermal vent shrimp Rimicaris hybisae 110291 EN Yuya Nagata Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Norio Miyamoto Institute for Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-Star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Keita Sato Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Nishimura Research Center for Bioscience and Nanoscience (CeBN), Research Institute for Marine Resources Utilization, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Yuki Tanioka School of Pharmaceutical Sciences, Okayama University Yuji Yamanaka School of Pharmaceutical Sciences, Okayama University Susumu Yoshizawa Atmosphere and Ocean Research Institute, The University of Tokyo Kuto Takahashi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kohei Obayashi Department of Biology, Graduate School of Science, Kobe University Hisao Tsukamoto Department of Biology, Graduate School of Science, Kobe University Ken Takai Institute for Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-Star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Hideyo Ohuchi Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takahiro Yamashita Department of Biophysics, Graduate School of Science, Kyoto University Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Unlike terrestrial environments, where humans reside, there is no sunlight in the deep sea. Instead, dim visible light from black-body radiation and bioluminescence illuminates hydrothermal vent areas in the deep sea. A deep-sea hydrothermal vent shrimp, Rimicaris hybisae, is thought to detect this dim light using its enlarged dorsal eye; however, the molecular basis of its photoreception remains unexplored. Here, we characterized the molecular properties of opsins, universal photoreceptive proteins in animals, found in R. hybisae. Transcriptomic analysis identified six opsins: three Gq-coupled opsins, one Opn3, one Opn5, and one peropsin. Functional analysis revealed that five of these opsins exhibited light-dependent G protein activity, whereas peropsin exhibited the ability to convert all-trans-retinal to 11-cis-retinal like photoisomerases. Notably, all the R. hybisae opsins, including Opn5, convergently show visible light sensitivity (around 457–517 nm), whereas most opsins categorized as Opn5 have been demonstrated to be UV sensitive. Mutational analysis revealed that the unique visible light sensitivity of R. hybisae Opn5 is achieved through the stabilization of a protonated Schiff base by a counterion residue at position 83 (Asp83), which differs from the position identified in other opsins. These findings suggest that the vent shrimp R. hybisae has adapted its photoreceptive devices to dim deep-sea hydrothermal light by selectively maintaining a repertoire of visible light–sensitive opsins, including the uniquely tuned Opn5. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1083-351X 301 4 2025 Roles of basic amino acid residues in substrate binding and transport of the light-driven anion pump Synechocystis halorhodopsin (SyHR) 108334 EN Masaki Nakama Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoyasu Noji Department of Applied Chemistry, The University of Tokyo Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Susumu Yoshizawa Atmosphere and Ocean Research Institute, The University of Tokyo Hiroshi Ishikita Department of Applied Chemistry, The University of Tokyo Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Microbial rhodopsins are photoreceptive seventransmembrane a-helical proteins, many of which function as ion transporters, primarily for small monovalent ions such as Na+, K+, Cl-, Br-, and I-. Synechocystis halorhodopsin (SyHR), identified from the cyanobacterium Synechocystis sp. PCC 7509, uniquely transports the polyatomic divalent SO42- inward, in addition to monovalent anions (Cl- and Br-). In this study, we conducted alanine-scanning mutagenesis on twelve basic amino acid residues to investigate the anion transport mechanism of SyHR. We quantitatively evaluated the Cl-and SO42- transport activities of the WT SyHR and its mutants. The results showed a strong correlation between the Cl-and SO42- transport activities among them (R = 0.94), suggesting a shared pathway for both anions. Notably, the R71A mutation selectively abolished SO42- transport activity while maintaining Cl- transport, whereas the H167A mutation significantly impaired both Cl-and SO42- transport. Furthermore, spectroscopic analysis revealed that the R71A mutant lost its ability to bind SO42- due to the absence of a positive charge, while the H167A mutant failed to accumulate the O intermediate during the photoreaction cycle (photocycle) due to reduced hydrophilicity. Additionally, computational analysis revealed the SO42- binding modes and clarified the roles of residues involved in its binding around the retinal chromophore. Based on these findings and previous structural information, we propose that the positive charge and hydrophilicity of Arg71 and His167 are crucial for the formation of the characteristic initial and transient anion-binding site of SyHR, enabling its unique ability to bind and transport both Cl-and SO42-. No potential conflict of interest relevant to this article was reported.

Pharmaceutical Society of Japan Acta Medica Okayama 0918-6158 47 10 2024 Molecular Diversity of Photosensitive Protein Opsins and Their High Potential for Optogenetic Applications 1600 1609 EN Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Because G protein coupled receptors (GPCRs) represent the largest family of drug targets in clinical trials, GPCR signaling cascades are closely related to various physiological phenomena, attracting significant attention in pharmaceutical science. Opsins (also known as animal rhodopsins) are photoreceptive proteins containing retinal as a chromophore, which function as GPCRs and underlie the molecular basis of photoreception in animals. Recently, opsins have been progressively applied in an innovative technology called optogenetics to regulate biological activities using light. A wide variety of opsins have been identified in metazoans and characterized at the molecular and physiological levels, providing a foundation for their optogenetic applications. In this review, I briefly introduce the diversity of opsins in terms of their molecular functions, including G protein selectivity and photoreaction properties. This diversity provides a significant advantage for optically manipulating a wide variety of GPCR signaling cascades with high temporal resolution. Additionally, I discuss the rich array of opsin-based optogenetic tools used to control various physiological processes and their potential as therapeutic tools for vision restoration. Based on the introduction, I expect that the optogenetic approach will offer powerful tools to provide valuable insights into the molecular mechanisms of various physiological phenomena and next-generation treatment options for diseases beyond the capacity of traditional drugs. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 1520-6106 128 27 2024 Bidirectional Optical Control of Proton Motive Force in Escherichia coli Using Microbial Rhodopsins 6509 6517 EN Kotaro Nakanishi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yoshiyuki Sowa Department of Frontier Bioscience and Research Center for Micro-Nano Technology, Hosei University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Proton (H+) motive force (PMF) serves as the energy source for the flagellar motor rotation, crucial for microbial motility. Here, to control PMF using light, we introduced light-driven inward and outward proton pump rhodopsins, RmXeR and AR3, into Escherichia coli. The motility of E. coli cells expressing RmXeR and AR3 significantly decreased and increased upon illumination, respectively. Tethered cell experiments revealed that, upon illumination, the torque of the flagellar motor decreased to nearly zero (28 pN nm) with RmXeR, while it increased to 1170 pN nm with AR3. These alterations in PMF correspond to +146 mV (RmXeR) and −140 mV (AR3), respectively. Thus, bidirectional optical control of PMF in E. coli was successfully achieved by using proton pump rhodopsins. This system holds a potential for enhancing our understanding of the roles of PMF in various biological functions. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2666-1667 3 1 2022 Expression of microbial rhodopsins in Escherichia coli and their extraction and purification using styrene-maleic acid copolymers 101046 EN Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Microbial rhodopsins are photoreceptive membrane proteins showing various light-dependent biological activities. Styrene-maleic acid (SMA) copolymers spontaneously form nanoscale lipid particles containing membrane proteins and associated lipids without detergent, and can be used to characterize membrane molecules. Here, we provide a protocol to functionally express a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin, and an unstable rhodopsin, Halobacterium salinarum sensory rhodopsin I, in Escherichia coli. We then describe the preparation of SMA and the extraction and purification of rhodopsin molecules using SMA. <br> For complete details on the use and execution of this protocol, please refer to Ueta et al. (2020). No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0006-291X 677 2023 Demonstration of iodide-dependent UVA-triggered growth inhibition in Saccharomyces cerevisiae cells and identification of its suppressive molecules 1 5 EN Ryota Ono Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Nozomu Saeki Research Core for Interdisciplinary Sciences, Okayama University Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hisao Moriya Research Core for Interdisciplinary Sciences, Okayama University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Upon white light illumination, the growth of the budding yeast Saccharomyces cerevisiae was extremely impaired only in the presence of iodide ions, but not fluoride, chloride and bromide ions. Action spectroscopy revealed that the maximum wavelength of the light is around at 373 nm, corresponding to the UVA region. Using a genetic approach, several genes, including OPY1, HEM1, and PAU11, were identified as suppressors of this growth inhibition. This iodide-dependent UVA-triggered growth inhibition method, along with its suppressive molecules, would be beneficial for understanding cell growth processes in eukaryotes and can be utilized for medium sterilization using UVA light. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2041-1723 14 1 2023 Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota 1730 EN Titouan Jaunet-Lahary Research Center for Computational Science, Institute for Molecular Science, National Institutes of Natural Sciences Tatsuro Shimamura Graduate School of Medicine, Kyoto University Masahiro Hayashi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Norimichi Nomura Graduate School of Medicine, Kyoto University Kouta Hirasawa Graduate School of Medicine, Kyoto University Tetsuya Shimizu RIKEN SPring-8 Center Masao Yamashita RIKEN SPring-8 Center Naotaka Tsutsumi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuta Suehiro School of Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takashi Tamura Graduate School of Environmental and Life Sciences, Okayama University Hiroko Iwanari Research Center for Advanced Science and Technology, The University of Tokyo Takao Hamakubo Research Center for Advanced Science and Technology, The University of Tokyo So Iwata Graduate School of Medicine, Kyoto University Kei-Ichi Okazaki Research Center for Computational Science, Institute for Molecular Science, National Institutes of Natural Sciences Teruhisa Hirai RIKEN SPring-8 Center Atsuko Yamashita Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis. No potential conflict of interest relevant to this article was reported.

nature portfolio Acta Medica Okayama 2045-2322 13 1 2023 A blue-shifted anion channelrhodopsin from the Colpodellida alga Vitrella brassicaformis 6974 EN Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shiho Kawanishi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Nishimura Research Center for Bioscience and Nanoscience (CeBN), Research Institute for Marine Resources Utilization, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Masumi Hasegawa Institute for Extra‑Cutting‑Edge Science and Technology Avant‑Garde Research (X‑Star) Shin Nakao Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuya Nagata Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Susumu Yoshizawa Atmosphere and Ocean Research Institute, The University of Tokyo Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Microbial rhodopsins, a family of photoreceptive membrane proteins containing the chromophore retinal, show a variety of light-dependent molecular functions. Channelrhodopsins work as light-gated ion channels and are widely utilized for optogenetics, which is a method for controlling neural activities by light. Since two cation channelrhodopsins were identified from the chlorophyte alga Chlamydomonas reinhardtii, recent advances in genomic research have revealed a wide variety of channelrhodopsins including anion channelrhodopsins (ACRs), describing their highly diversified molecular properties (e.g., spectral sensitivity, kinetics and ion selectivity). Here, we report two channelrhodopsin-like rhodopsins from the Colpodellida alga Vitrella brassicaformis, which are phylogenetically distinct from the known channelrhodopsins. Spectroscopic and electrophysiological analyses indicated that these rhodopsins are green- and blue-sensitive pigments (lambda(max) = similar to 550 and similar to 440 nm) that exhibit light-dependent ion channeling activities. Detailed electrophysiological analysis revealed that one of them works as a monovalent anion (Cl-, Br- and NO3-) channel and we named it V. brassicaformis anion channelrhodopsin-2, VbACR2. Importantly, the absorption maximum of VbACR2 (similar to 440 nm) is blue-shifted among the known ACRs. Thus, we identified the new blue-shifted ACR, which leads to the expansion of the molecular diversity of ACRs. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry (RSC) Acta Medica Okayama 1359-7345 59 49 2023 Development of light-induced disruptive liposomes (LiDL) as a photoswitchable carrier for intracellular substance delivery 7591 7594 EN Taichi Tsuneishi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Fumika Kubota Faculty of Pharmaceutical Sciences, Hokkaido University Hideyoshi Harashima Faculty of Pharmaceutical Sciences, Hokkaido University Yuma Yamada Faculty of Pharmaceutical Sciences, Hokkaido University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Light-driven inward proton pump rhodopsin RmXeR was embedded in pH-sensitive liposomes. Substance release from the proteoliposomes was observed following light illumination both in vitro and in cells, indicating the successful production of light-induced disruptive liposomes (LiDL). Thus, LiDL is a photoswitchable carrier utilized for intracellular substance delivery. No potential conflict of interest relevant to this article was reported.

Royal Society of Chemistry Acta Medica Okayama 2046-2069 13 11 2023 Correction: Convergent evolution of animal and microbial rhodopsins 7222 7224 EN Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Correction for 'Convergent evolution of animal and microbial rhodopsins' by Keiichi Kojima et al., RSC Adv., 2023, 13, 5367-5381, https://doi.org/10.1039/D2RA07073A. No potential conflict of interest relevant to this article was reported.

ROYAL SOC CHEMISTRY Acta Medica Okayama 2046-2069 13 8 2023 Convergent evolution of animal and microbial rhodopsins 5367 5381 EN Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Rhodopsins, a family of photoreceptive membrane proteins, contain retinal as a chromophore and were firstly identified as reddish pigments from frog retina in 1876. Since then, rhodopsin-like proteins have been identified mainly from animal eyes. In 1971, a rhodopsin-like pigment was discovered from the archaeon Halobacterium salinarum and named bacteriorhodopsin. While it was believed that rhodopsin- and bacteriorhodopsin-like proteins were expressed only in animal eyes and archaea, respectively, before the 1990s, a variety of rhodopsin-like proteins (called animal rhodopsins or opsins) and bacteriorhodopsin-like proteins (called microbial rhodopsins) have been progressively identified from various tissues of animals and microorganisms, respectively. Here, we comprehensively introduce the research conducted on animal and microbial rhodopsins. Recent analysis has revealed that the two rhodopsin families have common molecular properties, such as the protein structure (i.e., 7-transmembrane structure), retinal structure (i.e., binding ability to cis- and trans-retinal), color sensitivity (i.e., UV- and visible-light sensitivities), and photoreaction (i.e., triggering structural changes by light and heat), more than what was expected at the early stages of rhodopsin research. Contrastingly, their molecular functions are distinctively different (e.g., G protein-coupled receptors and photoisomerases for animal rhodopsins and ion transporters and phototaxis sensors for microbial rhodopsins). Therefore, based on their similarities and dissimilarities, we propose that animal and microbial rhodopsins have convergently evolved from their distinctive origins as multi-colored retinal-binding membrane proteins whose activities are regulated by light and heat but independently evolved for different molecular and physiological functions in the cognate organism. No potential conflict of interest relevant to this article was reported.

American Chemical Society Acta Medica Okayama 2470-1343 8 2023 Detection of Membrane Potential-Dependent Rhodopsin Fluorescence Using Low-Intensity Light Emitting Diode for Long-Term Imaging 4826 4834 EN Shiho Kawanishi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Atsushi Shibukawa Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayuki Sakamoto Department of Optical Neural and Molecular Physiology, Graduate School of Biostudies Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Faculty of Medicine, Dentistry and Pharmaceutical Sciences Microbial rhodopsin is a family of photoreceptive membrane proteins that commonly consist of a seven-transmembrane domain and a derivative of vitamin-A, retinal, as a chromophore. In 2011, archaeorhodopsin-3 (AR3) was shown to exhibit voltage-dependent fluorescence changes in mammalian cells. Since then, AR3 and its variants have been used as genetically encoded voltage indicators, in which mostly intense laser stimulation (1-1000 W/cm(2)) is used for the detection of dim fluorescence of rhodopsin, leading to high spatiotemporal resolution. However, intense laser stimulation potentially causes serious cell damage, particularly during long-term imaging over minutes. In this study, we present the successful detection of voltage-sensitive fluorescence of AR3 and its high fluorescence mutant Archon1 in a variety of mammalian cell lines using low-intensity light emitting diode stimulation (0.15 W/cm2) with long exposure time (500 ms). The detection system enables real-time imaging of drug-induced slow changes in voltage within the cells for minutes harmlessly and without fluorescence bleaching. Therefore, we demonstrate a method to quantitatively understand the dynamics of slow changes in membrane voltage on long time scales. No potential conflict of interest relevant to this article was reported.

Frontiers Media SA Acta Medica Okayama 2296-889X 8 2021 Exploring the Retinal Binding Cavity of Archaerhodopsin-3 by Replacing the Retinal Chromophore With a Dimethyl Phenylated Derivative 794948 EN Taichi Tsuneishi Laboratory of Biophysical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masataka Takahashi Laboratory of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masaki Tsujimura Department of Applied Chemistry, The University of Tokyo Keiichi Kojima Laboratory of Biophysical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroshi Ishikita Department of Applied Chemistry, The University of Tokyo Yasuo Takeuchi Laboratory of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Laboratory of Biophysical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Rhodopsins act as photoreceptors with their chromophore retinal (vitamin-A aldehyde) and they regulate light-dependent biological functions. Archaerhodopsin-3 (AR3) is an outward proton pump that has been widely utilized as a tool for optogenetics, a method for controlling cellular activity by light. To characterize the retinal binding cavity of AR3, we synthesized a dimethyl phenylated retinal derivative, (2E,4E,6E,8E)-9-(2,6-Dimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenal (DMP-retinal). QM/MM calculations suggested that DMP-retinal can be incorporated into the opsin of AR3 (archaeopsin-3, AO3). Thus, we introduced DMP-retinal into AO3 to obtain the non-natural holoprotein (AO3-DMP) and compared some molecular properties with those of AO3 with the natural A1-retinal (AO3-A1) or AR3. Light-induced pH change measurements revealed that AO3-DMP maintained slow outward proton pumping. Noteworthy, AO3-DMP had several significant changes in its molecular properties compared with AO3-A1 as follows; 1) spectroscopic measurements revealed that the absorption maximum was shifted from 556 to 508 nm and QM/MM calculations showed that the blue-shift was due to the significant increase in the HOMO-LUMO energy gap of the chromophore with the contribution of some residues around the chromophore, 2) time-resolved spectroscopic measurements revealed the photocycling rate was significantly decreased, and 3) kinetical spectroscopic measurements revealed the sensitivity of the chromophore binding Schiff base to attack by hydroxylamine was significantly increased. The QM/MM calculations show that a cavity space is present at the aromatic ring moiety in the AO3-DMP structure whereas it is absent at the corresponding beta-ionone ring moiety in the AO3-A1 structure. We discuss these alterations of the difference in interaction between the natural A1-retinal and the DMP-retinal with binding cavity residues. No potential conflict of interest relevant to this article was reported.

eLIFE SCIENCES PUBL LTD Acta Medica Okayama 2050-084X 10 2021 Proton transfer pathway in anion channelrhodopsin-1 e72264 EN Masaki Tsujimura Department of Applied Chemistry, The University of Tokyo Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shiho Kawanishi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hiroshi Ishikita Department of Applied Chemistry, The University of Tokyo Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 angstrom) and Glu68 (5.3 angstrom) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism. No potential conflict of interest relevant to this article was reported.

Pharmaceutical Society of Japan Acta Medica Okayama 0918-6158 44 10 2021 Microbial Rhodopsins as Multi-functional Photoreactive Membrane Proteins for Optogenetics 1357 1363 EN Shin Nakao Division of Pharmaceutical Sciences, Okayama University Keiichi Kojima Division of Pharmaceutical Sciences, Okayama University Yuki Sudo Division of Pharmaceutical Sciences, Okayama University In life science research, methods to control biological activities with stimuli such as light, heat, pressure and chemicals have been widely utilized to understand their molecular mechanisms. The knowledge obtained by those methods has built a basis for the development of medicinal products. Among those various stimuli, light has the advantage of a high spatiotemporal resolution that allows for the precise control of biological activities. Photoactive membrane protein rhodopsins from microorganisms (called microbial rhodopsins) absorb visible light and that light absorption triggers the trans–cis photoisomerization of the chromophore retinal, leading to various functions such as ion pumps, ion channels, transcriptional regulators and enzymes. In addition to their biological significance, microbial rhodopsins are widely utilized as fundamental molecular tools for optogenetics, a method to control biological activities by light. In this review, we briefly introduce the molecular basis of representative rhodopsin molecules and their applications for optogenetics. Based on those examples, we discuss the high potential of rhodopsin-based optogenetics tools for basic and clinical research in pharmaceutical sciences. No potential conflict of interest relevant to this article was reported.

Pharmaceutical Society of Japan Acta Medica Okayama 0031-6903 141 10 2021 光受容タンパク質・ロドプシンの生物物理化学研究 1155 1160 EN Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Opsins (also called animal rhodopsins) are universal photoreceptive proteins that provide the molecular basis of visual and nonvisual photoreception in animals, including humans. Opsins consist of seven helical a-transmembrane domains and use retinal, a derivative of vitamin A, as a chromophore. In many opsins, light absorption triggers photoisomerization from 11-cis retinal to all-trans retinal, resulting in activation via dynamic structural changes in the protein moiety. Activated opsins stimulate cognate trimeric G proteins to induce signal transduction cascades in cells. Recently, molecular and physiological analyses of diverse opsins have progressively advanced. This review introduces the molecular basis and physiological unctions of opsins. Based on the functions of opsins, I will discuss the potential of opsins as target molecules to treat and prevent visual and nonvisual diseases such as sleep disorder and depression. No potential conflict of interest relevant to this article was reported.