start-ver=1.4 cd-journal=joma no-vol=139 cd-vols= no-issue=12 article-no= start-page=4376 end-page=4389 dt-received= dt-revised= dt-accepted= dt-pub-year=2017 dt-pub=20170329 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Demonstration of a Light-Driven SO42- Transporter and Its Spectroscopic Characteristics. en-subtitle= kn-subtitle= en-abstract= kn-abstract= In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H+, Na+, and Cl-. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO42-, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl- or SO42-. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl- and SO42- with a subsequent secondary inward H+ movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl- and from 542 to 556 nm for SO42-, and the affinities of Cl- and SO42- were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO42-) transporter and report its spectroscopic characteristics. en-copyright= kn-copyright= en-aut-name=NihoAkiko en-aut-sei=Niho en-aut-mei=Akiko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=TsukamotoTakashi en-aut-sei=Tsukamoto en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KuriharaMarie en-aut-sei=Kurihara en-aut-mei=Marie kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=TaharaShinya en-aut-sei=Tahara en-aut-mei=Shinya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=NakajimaYu en-aut-sei=Nakajima en-aut-mei=Yu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=MizunoMisao en-aut-sei=Mizuno en-aut-mei=Misao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=KuramochiHikaru en-aut-sei=Kuramochi en-aut-mei=Hikaru kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=TaharaTahei en-aut-sei=Tahara en-aut-mei=Tahei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=MizutaniYasuhisa en-aut-sei=Mizutani en-aut-mei=Yasuhisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= affil-num=1 en-affil=Faculty of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil= Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil= Molecular Spectroscopy Laboratory, RIKEN kn-affil= affil-num=6 en-affil= Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=7 en-affil=Department of Chemistry, Graduate School of Science, Osaka University kn-affil= affil-num=8 en-affil=Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics kn-affil= affil-num=9 en-affil=Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics kn-affil= affil-num=10 en-affil=Department of Chemistry, Graduate School of Science, Osaka University kn-affil= affil-num=11 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=9 cd-vols= no-issue=1 article-no= start-page=7863 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20190527 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination en-subtitle= kn-subtitle= en-abstract= kn-abstract= Ion pumps and channels are responsible for a wide variety of biological functions. Ion pumps transport only one ion during each stimulus-dependent reaction cycle, whereas ion channels conduct a large number of ions during each cycle. Ion pumping rhodopsins such as archaerhodopsin-3 (Arch) are often utilized as light-dependent neural silencers in animals, but they require a high-density light illumination of around 1?mW/mm2. Recently, anion channelrhodopsins -1 and -2 (GtACR1 and GtACR2) were discovered as light-gated anion channels from the cryptophyte algae Guillardia theta. GtACRs are therefore expected to silence neural activity much more efficiently than Arch. In this study, we successfully expressed GtACRs in neurons of the nematode Caenorhabditis elegans (C. elegans) and quantitatively evaluated how potently GtACRs can silence neurons in freely moving C. elegans. The results showed that the light intensity required for GtACRs to cause locomotion paralysis was around 1??W/mm2, which is three orders of magnitude smaller than the light intensity required for Arch. As attractive features, GtACRs are less harmfulness to worms and allow stable neural silencing effects under long-term illumination. Our findings thus demonstrate that GtACRs possess a hypersensitive neural silencing activity in C. elegans and are promising tools for long-term neural silencing. en-copyright= kn-copyright= en-aut-name=YamanashiTaro en-aut-sei=Yamanashi en-aut-mei=Taro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=Maki Misayo en-aut-sei=Maki en-aut-mei= Misayo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=ShibukawaAtsushi en-aut-sei=Shibukawa en-aut-mei=Atsushi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=TsukamotoTakashi en-aut-sei=Tsukamoto en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ChowdhurySrikanta en-aut-sei=Chowdhury en-aut-mei=Srikanta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=YamanakaAkihiro en-aut-sei=Yamanaka en-aut-mei=Akihiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=TakagiShin en-aut-sei=Takagi en-aut-mei=Shin kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil= kn-affil= affil-num=7 en-affil= kn-affil= affil-num=8 en-affil= kn-affil= affil-num=9 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=58 cd-vols= no-issue=26 article-no= start-page=2934 end-page=2943 dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20190531 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Photochemical Characterization of a New Heliorhodopsin from the Gram-Negative Eubacterium Bellilinea caldifistulae (BcHeR) and Comparison with Heliorhodopsin-48C12 en-subtitle= kn-subtitle= en-abstract= kn-abstract= Many microorganisms express rhodopsins, pigmented membrane proteins capable of absorbing sunlight and harnessing that energy for important biological functions such as ATP synthesis and phototaxis. Microbial rhodopsins that have been discovered to date are categorized as type-1 rhodopsins. Interestingly, researchers have very recently unveiled a new microbial rhodopsin family named the heliorhodopsins, which are phylogenetically distant from type-1 rhodopsins. Among them, only heliorhodopsin-48C12 (HeR-48C12) from a Gram-positive eubacterium has been photochemically characterized [Pushkarev, A., et al. (2018) Nature 558, 595-599]. In this study, we photochemically characterize a purple-colored heliorhodopsin from Gram-negative eubacterium Bellilinea caldifistulae (BcHeR) as a second example and identify which properties are or are not conserved between BcHeR and HeR-48C12. A series of photochemical measurements revealed several conserved properties between them, including a visible absorption spectrum with a maximum at around 550 nm, the lack of ion-transport activity, and the existence of a second-order O-like intermediate during the photocycle that may activate an unidentified biological function. In contrast, as a property that is not conserved, although HeR-48C12 shows the light adaptation state of retinal, BcHeR showed the same retinal configuration under both dark- and light-adapted conditions. These comparisons of photochemical properties between BcHeR and HeR-48C12 are an important first step toward understanding the nature and functional role of heliorhodopsins. en-copyright= kn-copyright= en-aut-name=ShibukawaAtsushi en-aut-sei=Shibukawa en-aut-mei=Atsushi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NakajimaYu en-aut-sei=Nakajima en-aut-mei=Yu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=NishimuraYosuke en-aut-sei=Nishimura en-aut-mei=Yosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University kn-affil= affil-num=3 en-affil=Atmosphere and Ocean Research Institute , The University of Tokyo kn-affil= affil-num=4 en-affil=Atmosphere and Ocean Research Institute , The University of Tokyo kn-affil= affil-num=5 en-affil=Atmosphere and Ocean Research Institute , The University of Tokyo kn-affil= affil-num=6 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=59 cd-vols= no-issue=3 article-no= start-page=218 end-page=229 dt-received= dt-revised= dt-accepted= dt-pub-year=2019 dt-pub=20191209 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=The Unlimited Potential of Microbial Rhodopsins as Optical Tools en-subtitle= kn-subtitle= en-abstract= kn-abstract= Microbial rhodopsins, a photoactive membrane protein family, serve as fundamental tools for optogenetics, an innovative technology for controlling biological activities with light. Microbial rhodopsins are widely distributed in nature and have a wide variety of biological functions. Regardless of the many different known types of microbial rhodopsins, only a few of them have been used in optogenetics to control neural activity to understand neural networks. The efforts of our group have been aimed at identifying and characterizing novel rhodopsins from nature and also at engineering novel variant rhodopsins by rational design. On the basis of the molecular and functional characteristics of those novel rhodopsins, we have proposed new rhodopsin-based optogenetics tools to control not only neural activities but also "non-neural" activities. In this Perspective, we introduce the achievements and summarize future challenges in creating optogenetics tools using rhodopsins. The implementation of optogenetics deep inside an in vivo brain is the well-known challenge for existing rhodopsins. As a perspective to address this challenge, we introduce innovative optical illumination techniques using wavefront shaping that can reinforce the low light sensitivity of the rhodopsins and realize deep-brain optogenetics. The applications of our optogenetics tools could be extended to manipulate non-neural biological activities such as gene expression, apoptosis, energy production, and muscle contraction. We also discuss the potentially unlimited biotechnological applications of microbial rhodopsins in the future such as in photovoltaic devices and in drug delivery systems. We believe that advances in the field will greatly expand the potential uses of microbial rhodopsins as optical tools. en-copyright= kn-copyright= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ShibukawaAtsushi en-aut-sei=Shibukawa en-aut-mei=Atsushi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=10 cd-vols= no-issue=1 article-no= start-page=282 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20200114 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Vectorial Proton Transport Mechanism of RxR, a Phylogenetically Distinct and Thermally Stable Microbial Rhodopsin en-subtitle= kn-subtitle= en-abstract= kn-abstract=Rubrobacter xylanophilus rhodopsin (RxR) is a phylogenetically distinct and thermally stable seven-transmembrane protein that functions as a light-driven proton (H+) pump with the chromophore retinal. To characterize its vectorial proton transport mechanism, mutational and theoretical investigations were performed for carboxylates in the transmembrane region of RxR and the sequential proton transport steps were revealed as follows: (i) a proton of the retinylidene Schiff base (Lys209) is transferred to the counterion Asp74 upon formation of the blue-shifted M-intermediate in collaboration with Asp205, and simultaneously, a respective proton is released from the proton releasing group (Glu187/Glu197) to the extracellular side, (ii) a proton of Asp85 is transferred to the Schiff base during M-decay, (iii) a proton is taken up from the intracellular side to Asp85 during decay of the red-shifted O-intermediate. This ion transport mechanism of RxR provides valuable information to understand other ion transporters since carboxylates are generally essential for their functions. en-copyright= kn-copyright= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=UetaTetsuya en-aut-sei=Ueta en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NojiTomoyasu en-aut-sei=Noji en-aut-mei=Tomoyasu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=SaitoKeisuke en-aut-sei=Saito en-aut-mei=Keisuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KaneharaKanae en-aut-sei=Kanehara en-aut-mei=Kanae kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=IshikitaHiroshi en-aut-sei=Ishikita en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Faculty of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo kn-affil= affil-num=4 en-affil=Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo kn-affil= affil-num=5 en-affil=Faculty of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=7 en-affil=Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo kn-affil= affil-num=8 en-affil=Faculty of Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=Biochemistry kn-keyword=Biochemistry en-keyword=Biophysics kn-keyword=Biophysics END start-ver=1.4 cd-journal=joma no-vol=10 cd-vols= no-issue=1 article-no= start-page=20857 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2020 dt-pub=20201130 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift en-subtitle= kn-subtitle= en-abstract= kn-abstract=The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named 'schizorhodopsin (SzR)' has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pK(a)=12.3) is stabilised by the deprotonated counterion (presumably Asp184, pK(a)=3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral 'blue-shift' due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift. en-copyright= kn-copyright= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HasegawaMasumi en-aut-sei=Hasegawa en-aut-mei=Masumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=NakamaMasaki en-aut-sei=Nakama en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KuriharaMarie en-aut-sei=Kurihara en-aut-mei=Marie kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=KikukawaTakashi en-aut-sei=Kikukawa en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=3 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Faculty of Advanced Life Science, Hokkaido University kn-affil= affil-num=7 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=11 cd-vols= no-issue=1 article-no= start-page=14765 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20210720 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization en-subtitle= kn-subtitle= en-abstract= kn-abstract=Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pK(a)=3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins. en-copyright= kn-copyright= en-aut-name=KikuchiMasuzu en-aut-sei=Kikuchi en-aut-mei=Masuzu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NakaoShin en-aut-sei=Nakao en-aut-mei=Shin kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=KawanishiShiho en-aut-sei=Kawanishi en-aut-mei=Shiho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ShibukawaAtsushi en-aut-sei=Shibukawa en-aut-mei=Atsushi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=KikukawaTakashi en-aut-sei=Kikukawa en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Division of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Division of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Division of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=7 en-affil=Faculty of Advanced Life Science, Hokkaido University kn-affil= affil-num=8 en-affil=Division of Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=44 cd-vols= no-issue=10 article-no= start-page=1357 end-page=1363 dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=2021101 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Microbial Rhodopsins as Multi-functional Photoreactive Membrane Proteins for Optogenetics en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=NakaoShin en-aut-sei=Nakao en-aut-mei=Shin kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= affil-num=1 en-affil=Division of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Division of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Division of Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=rhodopsin kn-keyword=rhodopsin en-keyword=optogenetics kn-keyword=optogenetics en-keyword=retinal kn-keyword=retinal en-keyword=signal transduction kn-keyword=signal transduction END start-ver=1.4 cd-journal=joma no-vol=10 cd-vols= no-issue= article-no= start-page=e72264 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20211221 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Proton transfer pathway in anion channelrhodopsin-1 en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=TsujimuraMasaki en-aut-sei=Tsujimura en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KawanishiShiho en-aut-sei=Kawanishi en-aut-mei=Shiho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=IshikitaHiroshi en-aut-sei=Ishikita en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Department of Applied Chemistry, The University of Tokyo kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Applied Chemistry, The University of Tokyo kn-affil= END start-ver=1.4 cd-journal=joma no-vol=8 cd-vols= no-issue= article-no= start-page=794948 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2021 dt-pub=20211220 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Exploring the Retinal Binding Cavity of Archaerhodopsin-3 by Replacing the Retinal Chromophore With a Dimethyl Phenylated Derivative en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=TsuneishiTaichi en-aut-sei=Tsuneishi en-aut-mei=Taichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=TakahashiMasataka en-aut-sei=Takahashi en-aut-mei=Masataka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=TsujimuraMasaki en-aut-sei=Tsujimura en-aut-mei=Masaki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=IshikitaHiroshi en-aut-sei=Ishikita en-aut-mei=Hiroshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=TakeuchiYasuo en-aut-sei=Takeuchi en-aut-mei=Yasuo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= affil-num=1 en-affil=Laboratory of Biophysical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Laboratory of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Department of Applied Chemistry, The University of Tokyo kn-affil= affil-num=4 en-affil=Laboratory of Biophysical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Applied Chemistry, The University of Tokyo kn-affil= affil-num=6 en-affil=Laboratory of Synthetic and Medicinal Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=7 en-affil=Laboratory of Biophysical Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=retinal kn-keyword=retinal en-keyword=rhodopsin kn-keyword=rhodopsin en-keyword=proton pump kn-keyword=proton pump en-keyword=derivative kn-keyword=derivative en-keyword=photoreceptor kn-keyword=photoreceptor END start-ver=1.4 cd-journal=joma no-vol=8 cd-vols= no-issue= article-no= start-page=4826 end-page=4834 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230125 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Detection of Membrane Potential-Dependent Rhodopsin Fluorescence Using Low-Intensity Light Emitting Diode for Long-Term Imaging en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KawanishiShiho en-aut-sei=Kawanishi en-aut-mei=Shiho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=ShibukawaAtsushi en-aut-sei=Shibukawa en-aut-mei=Atsushi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=SakamotoMasayuki en-aut-sei=Sakamoto en-aut-mei=Masayuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Department of Optical Neural and Molecular Physiology, Graduate School of Biostudies kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Faculty of Medicine, Dentistry and Pharmaceutical Sciences kn-affil= END start-ver=1.4 cd-journal=joma no-vol=13 cd-vols= no-issue=8 article-no= start-page=5367 end-page=5381 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230213 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Convergent evolution of animal and microbial rhodopsins en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= affil-num=1 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=13 cd-vols= no-issue=11 article-no= start-page=7222 end-page=7224 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230306 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Correction: Convergent evolution of animal and microbial rhodopsins en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= affil-num=1 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=71 cd-vols= no-issue=2 article-no= start-page=154 end-page=164 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Identification of a Functionally Efficient and Thermally Stable Outward Sodium-Pumping Rhodopsin (BeNaR) from a Thermophilic Bacterium en-subtitle= kn-subtitle= en-abstract= kn-abstract=Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+-pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524?nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+-pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools. en-copyright= kn-copyright= en-aut-name=KuriharaMarie en-aut-sei=Kurihara en-aut-mei=Marie kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ThielVera en-aut-sei=Thiel en-aut-mei=Vera kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=TakahashiHirona en-aut-sei=Takahashi en-aut-mei=Hirona kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=WardDavid M. en-aut-sei=Ward en-aut-mei=David M. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=BryantDonald A. en-aut-sei=Bryant en-aut-mei=Donald A. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SakaiMakoto en-aut-sei=Sakai en-aut-mei=Makoto kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Department of Biological Sciences, Tokyo Metropolitan University kn-affil= affil-num=3 en-affil=Department of Chemistry, Graduate School of Science, Okayama University of Science kn-affil= affil-num=4 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Land Resources and Environmental Sciences, Montana State University kn-affil= affil-num=6 en-affil=Department of Biochemistry and Molecular Biology, The Pennsylvania State University kn-affil= affil-num=7 en-affil=Department of Chemistry, Graduate School of Science, Okayama University of Science kn-affil= affil-num=8 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=9 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=rhodopsin kn-keyword=rhodopsin en-keyword=ion transport kn-keyword=ion transport en-keyword=retinal kn-keyword=retinal en-keyword=isomerization kn-keyword=isomerization en-keyword=optogenetics kn-keyword=optogenetics END start-ver=1.4 cd-journal=joma no-vol=59 cd-vols= no-issue=49 article-no= start-page=7591 end-page=7594 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=2023 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Development of light-induced disruptive liposomes (LiDL) as a photoswitchable carrier for intracellular substance delivery en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=TsuneishiTaichi en-aut-sei=Tsuneishi en-aut-mei=Taichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KubotaFumika en-aut-sei=Kubota en-aut-mei=Fumika kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HarashimaHideyoshi en-aut-sei=Harashima en-aut-mei=Hideyoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=YamadaYuma en-aut-sei=Yamada en-aut-mei=Yuma kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Faculty of Pharmaceutical Sciences, Hokkaido University kn-affil= affil-num=4 en-affil=Faculty of Pharmaceutical Sciences, Hokkaido University kn-affil= affil-num=5 en-affil=Faculty of Pharmaceutical Sciences, Hokkaido University kn-affil= affil-num=6 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=13 cd-vols= no-issue=1 article-no= start-page=6974 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230428 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=A blue-shifted anion channelrhodopsin from the Colpodellida alga Vitrella brassicaformis en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=KawanishiShiho en-aut-sei=Kawanishi en-aut-mei=Shiho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=NishimuraYosuke en-aut-sei=Nishimura en-aut-mei=Yosuke kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=HasegawaMasumi en-aut-sei=Hasegawa en-aut-mei=Masumi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=NakaoShin en-aut-sei=Nakao en-aut-mei=Shin kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=NagataYuya en-aut-sei=Nagata en-aut-mei=Yuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= affil-num=1 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=3 en-affil=Research Center for Bioscience and Nanoscience (CeBN), Research Institute for Marine Resources Utilization, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) kn-affil= affil-num=4 en-affil=Institute for Extra?Cutting?Edge Science and Technology Avant?Garde Research (X?Star) kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=6 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=7 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=8 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=14 cd-vols= no-issue=1 article-no= start-page=1730 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230403 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=Jaunet-LaharyTitouan en-aut-sei=Jaunet-Lahary en-aut-mei=Titouan kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ShimamuraTatsuro en-aut-sei=Shimamura en-aut-mei=Tatsuro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=HayashiMasahiro en-aut-sei=Hayashi en-aut-mei=Masahiro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=NomuraNorimichi en-aut-sei=Nomura en-aut-mei=Norimichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=HirasawaKouta en-aut-sei=Hirasawa en-aut-mei=Kouta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=ShimizuTetsuya en-aut-sei=Shimizu en-aut-mei=Tetsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=YamashitaMasao en-aut-sei=Yamashita en-aut-mei=Masao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=TsutsumiNaotaka en-aut-sei=Tsutsumi en-aut-mei=Naotaka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=SuehiroYuta en-aut-sei=Suehiro en-aut-mei=Yuta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=TamuraTakashi en-aut-sei=Tamura en-aut-mei=Takashi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=IwanariHiroko en-aut-sei=Iwanari en-aut-mei=Hiroko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=HamakuboTakao en-aut-sei=Hamakubo en-aut-mei=Takao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= en-aut-name=IwataSo en-aut-sei=Iwata en-aut-mei=So kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=15 ORCID= en-aut-name=OkazakiKei-Ichi en-aut-sei=Okazaki en-aut-mei=Kei-Ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=16 ORCID= en-aut-name=HiraiTeruhisa en-aut-sei=Hirai en-aut-mei=Teruhisa kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=17 ORCID= en-aut-name=YamashitaAtsuko en-aut-sei=Yamashita en-aut-mei=Atsuko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=18 ORCID= affil-num=1 en-affil=Research Center for Computational Science, Institute for Molecular Science, National Institutes of Natural Sciences kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=3 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=6 en-affil=RIKEN SPring-8 Center kn-affil= affil-num=7 en-affil=RIKEN SPring-8 Center kn-affil= affil-num=8 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=9 en-affil=School of Pharmaceutical Sciences, Okayama University kn-affil= affil-num=10 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=11 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=12 en-affil=Graduate School of Environmental and Life Sciences, Okayama University kn-affil= affil-num=13 en-affil=Research Center for Advanced Science and Technology, The University of Tokyo kn-affil= affil-num=14 en-affil=Research Center for Advanced Science and Technology, The University of Tokyo kn-affil= affil-num=15 en-affil=Graduate School of Medicine, Kyoto University kn-affil= affil-num=16 en-affil=Research Center for Computational Science, Institute for Molecular Science, National Institutes of Natural Sciences kn-affil= affil-num=17 en-affil=RIKEN SPring-8 Center kn-affil= affil-num=18 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=677 cd-vols= no-issue= article-no= start-page=1 end-page=5 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20231015 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Demonstration of iodide-dependent UVA-triggered growth inhibition in Saccharomyces cerevisiae cells and identification of its suppressive molecules en-subtitle= kn-subtitle= en-abstract= kn-abstract=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. en-copyright= kn-copyright= en-aut-name=OnoRyota en-aut-sei=Ono en-aut-mei=Ryota kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=SaekiNozomu en-aut-sei=Saeki en-aut-mei=Nozomu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=MoriyaHisao en-aut-sei=Moriya en-aut-mei=Hisao kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Research Core for Interdisciplinary Sciences, Okayama University kn-affil= affil-num=3 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=4 en-affil=Research Core for Interdisciplinary Sciences, Okayama University kn-affil= affil-num=5 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=UVA kn-keyword=UVA en-keyword=Saccharomyces cerevisiae kn-keyword=Saccharomyces cerevisiae en-keyword=Iodide kn-keyword=Iodide en-keyword=Growth inhibition kn-keyword=Growth inhibition en-keyword=Suppressive molecule kn-keyword=Suppressive molecule END start-ver=1.4 cd-journal=joma no-vol=3 cd-vols= no-issue=1 article-no= start-page=101046 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2022 dt-pub=20220318 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Expression of microbial rhodopsins in Escherichia coli and their extraction and purification using styrene-maleic acid copolymers en-subtitle= kn-subtitle= en-abstract= kn-abstract=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.
For complete details on the use and execution of this protocol, please refer to Ueta et al. (2020). en-copyright= kn-copyright= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= END start-ver=1.4 cd-journal=joma no-vol=71 cd-vols= no-issue=2 article-no= start-page=154 end-page=164 dt-received= dt-revised= dt-accepted= dt-pub-year=2023 dt-pub=20230201 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Identification of a Functionally Efficient and Thermally Stable Outward Sodium-Pumping Rhodopsin (BeNaR) from a Thermophilic Bacterium en-subtitle= kn-subtitle= en-abstract= kn-abstract=Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+-pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524?nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+-pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools. en-copyright= kn-copyright= en-aut-name=KuriharaMarie en-aut-sei=Kurihara en-aut-mei=Marie kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=ThielVera en-aut-sei=Thiel en-aut-mei=Vera kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=TakahashiHirona en-aut-sei=Takahashi en-aut-mei=Hirona kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=KojimaKeiichi en-aut-sei=Kojima en-aut-mei=Keiichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=WardDavid M. en-aut-sei=Ward en-aut-mei=David M. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=BryantDonald A. en-aut-sei=Bryant en-aut-mei=Donald A. kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=SakaiMakoto en-aut-sei=Sakai en-aut-mei=Makoto kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=YoshizawaSusumu en-aut-sei=Yoshizawa en-aut-mei=Susumu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=SudoYuki en-aut-sei=Sudo en-aut-mei=Yuki kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= affil-num=1 en-affil=Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=2 en-affil=Department of Biological Sciences, Tokyo Metropolitan University kn-affil= affil-num=3 en-affil=Department of Chemistry, Graduate School of Science, Okayama University of Science kn-affil= affil-num=4 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= affil-num=5 en-affil=Department of Land Resources and Environmental Sciences, Montana State University kn-affil= affil-num=6 en-affil=Department of Biochemistry and Molecular Biology, The Pennsylvania State University kn-affil= affil-num=7 en-affil=Department of Chemistry, Graduate School of Science, Okayama University of Science kn-affil= affil-num=8 en-affil=Atmosphere and Ocean Research Institute, The University of Tokyo kn-affil= affil-num=9 en-affil=Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University kn-affil= en-keyword=rhodopsin kn-keyword=rhodopsin en-keyword=ion transport kn-keyword=ion transport en-keyword=retinal kn-keyword=retinal en-keyword=isomerization kn-keyword=isomerization en-keyword=optogenetics kn-keyword=optogenetics END