Research activity information

• A repertoire of visible light-sensitive opsins in the deep-sea hydrothermal vent shrimp Rimicaris hybisae

  Yuya Nagata, Norio Miyamoto, Keita Sato, Yosuke Nishimura, Yuki Tanioka, Yuji Yamanaka, Susumu Yoshizawa, Kuto Takahashi, Kohei Obayashi, Hisao Tsukamoto, Ken Takai, Hideyo Ohuchi, Takahiro Yamashita, Yuki Sudo, Keiichi Kojima

  Elsevier BV, May 2025, Journal of Biological Chemistry, 110291 - 110291, English

  [Refereed]

  Scientific journal


• Molecular basis underlying the specificity of an antagonist AA92593 for mammalian melanopsins

  Kohei Obayashi, Ruisi Zou, Tomoki Kawaguchi, Toshifumi Mori, Hisao Tsukamoto

  Elsevier BV, May 2025, Journal of Biological Chemistry, 301(5) (5), 108461 - 108461, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Characterization and Engineering of a Blue-Sensitive, Gi/o-Biased, and Bistable Ciliary Opsin from a Fan Worm

  Sachiko Fukuzawa, Tomoki Kawaguchi, Takushi Shimomura, Yoshihiro Kubo, Hisao Tsukamoto

  American Chemical Society (ACS), Feb. 2025, Biochemistry, 64(5) (5), 1020 - 1031, English

  [Refereed]

  Scientific journal


• Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

  Kentaro Fukuta, Dai-Ichiro Kato, Juri Maeda, Atsuhiro Tsuruta, Hirobumi Suzuki, Yukio Nagano, Hisao Tsukamoto, Kazuki Niwa, Makoto Terauchi, Atsushi Toyoda, Asao Fujiyama, Hideki Noguchi

  The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

  Apr. 2024, DNA research : an international journal for rapid publication of reports on genes and genomes, 31(2) (2), English, International magazine

  [Refereed]

  Scientific journal


• A self-inactivating invertebrate opsin optically drives biased signaling toward Gβγ-dependent ion channel modulation.

  Hisao Tsukamoto, Yoshihiro Kubo

  Animal opsins, light-sensitive G protein-coupled receptors, have been used for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Gα and Gβγ subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Gα- and Gβγ-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Gα and Gβγ Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin, Platynereis c-opsin1, drives biased signaling for Gβγ-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between Gβγ-dependent and Gα-dependent responses. The opsin-induced transient Gi/o activation preferentially causes activation of the kinetically fast Gβγ-dependent GIRK channels rather than slower Gi/oα-dependent adenylyl cyclase inhibition. Although similar Gβγ-biased signaling properties were observed in a self-inactivating vertebrate visual pigment, Platynereis c-opsin1 requires fewer retinal molecules to evoke cellular responses. Furthermore, the Gβγ-biased signaling properties of Platynereis c-opsin1 are enhanced by genetically fusing with RGS8 protein, which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for Gβγ-dependent ion channel modulation.

  May 2023, Proceedings of the National Academy of Sciences of the United States of America, 120(21) (21), e2301269120, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Optogenetic Modulation of Ion Channels by Photoreceptive Proteins.

  Hisao Tsukamoto, Yuji Furutani

  In these 15 years, researches to control cellular responses by light have flourished dramatically to establish "optogenetics" as a research field. In particular, light-dependent excitation/inhibition of neural cells using channelrhodopsins or other microbial rhodopsins is the most powerful and the most widely used optogenetic technique. New channelrhodopsin-based optogenetic tools having favorable characteristics have been identified from a wide variety of organisms or created through mutagenesis. Despite the great efforts, some neuronal activities are still hard to be manipulated by the channelrhodopsin-based tools, indicating that complementary approaches are needed to make optogenetics more comprehensive. One of the feasible and complementary approaches is optical control of ion channels using photoreceptive proteins other than channelrhodopsins. In particular, animal opsins can modulate various ion channels via light-dependent G protein activation. In this chapter, we summarize how such alternative optogenetic tools work and they will be improved.

  2021, Advances in experimental medicine and biology, 1293, 73 - 88, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Session 2SFA-the symposium "Elucidation of biological functions by optical control" on BSJ2019 at Miyazaki, Japan.

  Hisao Tsukamoto, Yoshinori Shichida

  The symposium "Elucidation of biological functions by optical control" was held during the 57th annual meeting of the Biophysical Society of Japan (BSJ2019) at Miyazaki, Japan. In this commentary, we introduce invited speakers of this symposium and summarized their research topics.

  Feb. 2020, Biophysical reviews, English, International magazine

  [Refereed]

  Symposium


• The counterion-retinylidene Schiff base interaction of an invertebrate rhodopsin rearranges upon light activation.

  Nagata T, Koyanagi M, Tsukamoto H, Mutt E, Schertler GFX, Deupi X, Terakita A

  Animals sense light using photosensitive proteins-rhodopsins-containing a chromophore-retinal-that intrinsically absorbs in the ultraviolet. Visible light-sensitivity depends primarily on protonation of the retinylidene Schiff base (SB), which requires a negatively-charged amino acid residue-counterion-for stabilization. Little is known about how the most common counterion among varied rhodopsins, Glu181, functions. Here, we demonstrate that in a spider visual rhodopsin, orthologue of mammal melanopsins relevant to circadian rhythms, the Glu181 counterion functions likely by forming a hydrogen-bonding network, where Ser186 is a key mediator of the Glu181-SB interaction. We also suggest that upon light activation, the Glu181-SB interaction rearranges while Ser186 changes its contribution. This is in contrast to how the counterion of vertebrate visual rhodopsins, Glu113, functions, which forms a salt bridge with the SB. Our results shed light on the molecular mechanisms of visible light-sensitivity relevant to invertebrate vision and vertebrate non-visual photoreception.

  2019, Communications biology, 2, 180 - 180, English, International magazine

  [Refereed]

  Scientific journal


• A Go-type opsin mediates the shadow reflex in the annelid Platynereis dumerilii

  Thomas Ayers, Hisao Tsukamoto, Martin Gühmann, Vinoth Babu Veedin Rajan, Kristin Tessmar-Raible

  BioMed Central Ltd., Apr. 2018, BMC Biology, 16(1) (1), 41, English

  [Refereed]

  Scientific journal


• Structural properties determining low K affinity of the selectivity filter in the TWIK1 K channel

  Hisao Tsukamoto, Masahiro Higashi, Hideyoshi Motoki, Hiroki Watanabe, Christian Ganser, Koichi Nakajo, Yoshihiro Kubo, Takayuki Uchihashi, Yuji Furutani

  American Society for Biochemistry and Molecular Biology Inc., 2018, Journal of Biological Chemistry, 293(18) (18), 6969 - 6984, English

  [Refereed]

  Scientific journal


• A ciliary opsin in the brain of a marine annelid zooplankton is ultraviolet-sensitive, and the sensitivity is tuned by a single amino acid residue

  Hisao Tsukamoto, I-Shan Chen, Yoshihiro Kubo, Yuji Furutani

  Aug. 2017, JOURNAL OF BIOLOGICAL CHEMISTRY, 292(31) (31), 12971 - 12980, English

  [Refereed]

  Scientific journal


• Structural insights into the nucleotide base specificity of P2X receptors

  Go Kasuya, Yuichiro Fujiwara, Hisao Tsukamoto, Satoshi Morinaga, Satoshi Ryu, Kazushige Touhara, Ryuichiro Ishitani, Yuji Furutani, Motoyuki Hattori, Osamu Nureki

  Nature Publishing Group, Mar. 2017, Scientific Reports, 7, 45208, English

  [Refereed]

  Scientific journal


• Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

  Hisao Tsukamoto, Yoshihiro Kubo, David L. Farrens, Mitsumasa Koyanagi, Akihisa Terakita, Yuji Furutani

  Nov. 2015, JOURNAL OF BIOLOGICAL CHEMISTRY, 290(45) (45), 27176 - 27187, English

  [Refereed]

  Scientific journal


• Activation of Transducin by Bistable Pigment Parapinopsin in the Pineal Organ of Lower Vertebrates

  Emi Kawano-Yamashita, Mitsumasa Koyanagi, Seiji Wada, Hisao Tsukamoto, Takashi Nagata, Akihisa Terakita

  Oct. 2015, PLOS ONE, 10(10) (10), e0141280, English

  [Refereed]

  Scientific journal


• Diversification of non-visual photopigment parapinopsin in spectral sensitivity for diverse pineal functions.

  Mitsumasa Koyanagi, Seiji Wada, Emi Kawano-Yamashita, Yuichiro Hara, Shigehiro Kuraku, Shigeaki Kosaka, Koichi Kawakami, Satoshi Tamotsu, Hisao Tsukamoto, Yoshinori Shichida, Akihisa Terakita

  BACKGROUND: Recent genome projects of various animals have uncovered an unexpectedly large number of opsin genes, which encode protein moieties of photoreceptor molecules, in most animals. In visual systems, the biological meanings of this diversification are clear; multiple types of visual opsins with different spectral sensitivities are responsible for color vision. However, the significance of the diversification of non-visual opsins remains uncertain, in spite of the importance of understanding the molecular mechanism and evolution of varied non-visual photoreceptions. RESULTS: Here, we investigated the diversification of the pineal photopigment parapinopsin, which serves as the UV-sensitive photopigment for the pineal wavelength discrimination in the lamprey, linking it with other pineal photoreception. Spectroscopic analyses of the recombinant pigments of the two teleost parapinopsins PP1 and PP2 revealed that PP1 is a UV-sensitive pigment, similar to lamprey parapinopsin, but PP2 is a blue-sensitive pigment, with an absorption maximum at 460-480 nm, showing the diversification of non-visual pigment with respect to spectral sensitivity. We also found that PP1 and PP2 exhibit mutually exclusive expressions in the pineal organs of three teleost species. By using transgenic zebrafish in which these parapinopsin-expressing cells are labeled, we found that PP1-expressing cells basically possess neuronal processes, which is consistent with their involvement in wavelength discrimination. Interestingly, however, PP2-expressing cells rarely possess neuronal processes, raising the possibility that PP2 could be involved in non-neural responses rather than neural responses. Furthermore, we found that PP2-expressing cells contain serotonin and aanat2, the key enzyme involved in melatonin synthesis from serotonin, whereas PP1-expressing cells do not contain either, suggesting that blue-sensitive PP2 is instead involved in light-regulation of melatonin secretion. CONCLUSIONS: In this paper, we have clearly shown the different molecular properties of duplicated non-visual opsins by demonstrating the diversification of parapinopsin with respect to spectral sensitivity. Moreover, we have shown a plausible link between the diversification and its physiological impact by discovering a strong candidate for the underlying pigment in light-regulated melatonin secretion in zebrafish; the diversification could generate a new contribution of parapinopsin to pineal photoreception. Current findings could also provide an opportunity to understand the "color" preference of non-visual photoreception.

  Sep. 2015, BMC biology, 13, 73 - 73, English, International magazine

  [Refereed]


• Chimeras of Channelrhodopsin-1 and-2 from Chlamydomonas reinhardtii Exhibit Distinctive Light-induced Structural Changes from Channelrhodopsin-2

  Asumi Inaguma, Hisao Tsukamoto, Hideaki E. Kato, Tetsunari Kimura, Toru Ishizuka, Satomi Oishi, Hiromu Yawo, Osamu Nureki, Yuji Furutani

  May 2015, JOURNAL OF BIOLOGICAL CHEMISTRY, 290(18) (18), 11623 - 11634, English

  [Refereed]

  Scientific journal


• Distribution of Mammalian-Like Melanopsin in Cyclostome Retinas Exhibiting a Different Extent of Visual Functions

  Lanfang Sun, Emi Kawano-Yamashita, Takashi Nagata, Hisao Tsukamoto, Yuji Furutani, Mitsumasa Koyanagi, Akihisa Terakita

  Sep. 2014, PLOS ONE, 9(9) (9), e108209, English

  [Refereed]

  Scientific journal


• A Constitutively Activating Mutation Alters the Dynamics and Energetics of a Key Conformational Change in a Ligand-free G Protein-coupled Receptor

  Hisao Tsukamoto, David L. Farrens

  Sep. 2013, JOURNAL OF BIOLOGICAL CHEMISTRY, 288(39) (39), 28207 - 28216, English

  [Refereed]

  Scientific journal


• Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue

  Mitsumasa Koyanagi, Eiichiro Takada, Takashi Nagata, Hisao Tsukamoto, Akihisa Terakita

  Mar. 2013, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110(13) (13), 4998 - 5003, English

  [Refereed]

  Scientific journal


• Photochemical Nature of Parietopsin

  Kazumi Sakai, Yasushi Imamoto, Chih-Ying Su, Hisao Tsukamoto, Takahiro Yamashita, Akihisa Terakita, King-Wai Yau, Yoshinori Shichida

  Mar. 2012, BIOCHEMISTRY, 51(9) (9), 1933 - 1941, English

  [Refereed]

  Scientific journal


• Depth Perception from Image Defocus in a Jumping Spider

  Takashi Nagata, Mitsumasa Koyanagi, Hisao Tsukamoto, Shinjiro Saeki, Kunio Isono, Yoshinori Shichida, Fumio Tokunaga, Michiyo Kinoshita, Kentaro Arikawa, Akihisa Terakita

  Jan. 2012, SCIENCE, 335(6067) (6067), 469 - 471, English

  [Refereed]

  Scientific journal


• Rhodopsin in Nanodiscs Has Native embrane-like Photointermediates

  Hisao Tsukamoto, Istvan Szundi, James W. Lewis, David L. Farrens, David S. Kliger

  Jun. 2011, BIOCHEMISTRY, 50(22) (22), 5086 - 5091, English

  [Refereed]

  Scientific journal


• Monomeric Rhodopsin Is the Minimal Functional Unit Required for Arrestin Binding

  Hisao Tsukamoto, Abhinav Sinha, Mark DeWitt, David L. Farrens

  Jun. 2010, JOURNAL OF MOLECULAR BIOLOGY, 399(3) (3), 501 - 511, English

  [Refereed]

  Scientific journal


• A Pivot between Helices V and VI near the Retinal-binding Site Is Necessary for Activation in Rhodopsins

  Hisao Tsukamoto, Akihisa Terakita, Yoshinori Shichida

  Mar. 2010, JOURNAL OF BIOLOGICAL CHEMISTRY, 285(10) (10), 7351 - 7357, English

  [Refereed]

  Scientific journal


• 3P275 Investigation on a relationship of spectral characteristics of the rhodopsins and depth perception mechanism in a jumping spider(Photobiology: Vision & Photoreception,The 48th Annual Meeting of the Biophysical Society of Japan)

  Nagata Takashi, Koyanagi Mitsumasa, Tsukamoto Hisao, Isono Kunio, Arikawa Kentaro, Terakita Akihisa

  The Biophysical Society of Japan General Incorporated Association, 2010, Seibutsu Butsuri, 50(2) (2), S194, English


• Identification and characterization of a protostome homologue of peropsin from a jumping spider

  Takashi Nagata, Mitsumasa Koyanagi, Hisao Tsukamoto, Akihisa Terakita

  Jan. 2010, JOURNAL OF COMPARATIVE PHYSIOLOGY A-NEUROETHOLOGY SENSORY NEURAL AND BEHAVIORAL PHYSIOLOGY, 196(1) (1), 51 - 59, English

  [Refereed]

  Scientific journal


• Diversity and functional properties of bistable pigments

  Hisao Tsukamoto, Akihisa Terakita

  2010, PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 9(11) (11), 1435 - 1443, English

  [Refereed]


• The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

  Hisao Tsukamoto, David L. Farrens, Mitsumasa Koyanagi, Akihisa Terakita

  Jul. 2009, JOURNAL OF BIOLOGICAL CHEMISTRY, 284(31) (31), 20676 - 20683, English

  [Refereed]

  Scientific journal


• Report of 4th Asia Oceania Conference on Photobiology (AOCP)

  Hisao TSUKAMOTO

  Biophysical Society of Japan, 2009, Seibutsu Butsuri, 49(2) (2), 098 - 099, Japanese

  [Refereed][Invited]


• Jellyfish vision starts with cAMP signaling mediated by opsin-G(s) cascade

  Mitsumasa Koyanagi, Kosuke Takano, Hisao Tsukamoto, Kohzoh Ohtsu, Fumio Tokunaga, Akihisa Terakita

  Oct. 2008, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 105(40) (40), 15576 - 15580, English

  [Refereed]

  Scientific journal


• Expression and comparative characterization of Gq-coupled invertebrate visual pigments and melanopsin

  Akihisa Terakita, Hisao Tsukamoto, Mitsumasa Koyanagi, Michio Sugahara, Takahiro Yamashita, Yoshinori Shichida

  May 2008, JOURNAL OF NEUROCHEMISTRY, 105(3) (3), 883 - 890, English

  [Refereed]

  Scientific journal


• 1P-272 Photoreaction of parietopsin(The 46th Annual Meeting of the Biophysical Society of Japan)

  Sakai Kazumi, Imamoto Yasushi, Yamashita Takahiro, Tsukamoto Hisao, Terakita Akihisa, Su Chih-Ying, Yau King-Wai, Shichida Yoshinori

  The Biophysical Society of Japan General Incorporated Association, 2008, Seibutsu Butsuri, 48, S64, English


• 2P334 Mutational analyses of amino acid-interactions around the retinal Schiff base in the invertebrate rhodopsin(Photobiology-vision and photoreception,Oral Presentations)

  Nagata Takashi, Tsukamoto Hisao, Koyanagi Mitsumasa, Terakita Akihisa

  The Biophysical Society of Japan General Incorporated Association, 2007, Seibutsu Butsuri, 47, S196, English


• 2P335 Detection of structural changes involved in activation of diverged rhodopsins using site-directed fluorescence labeling(Photobiology-vision and photoreception,Oral Presentations)

  Tsukamoto Hisao, Koyanagi Mitsumasa, Terakita Akihisa

  The Biophysical Society of Japan General Incorporated Association, 2007, Seibutsu Butsuri, 47, S196, English


• Cephalochordate melanopsin: Evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells

  M Koyanagi, K Kubokawa, H Tsukamoto, Y Shichida, A Terakita

  Jun. 2005, CURRENT BIOLOGY, 15(11) (11), 1065 - 1069, English

  [Refereed]

  Scientific journal


• A rhodopsin exhibiting binding ability to agonist all-trans-retinal

  H Tsukamoto, A Terakita, Y Shichida

  May 2005, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(18) (18), 6303 - 6308, English

  [Refereed]

  Scientific journal


• 1P278 Comparative analyses of cephalochordate melanopsin with Gq-coupled invertebrate rhodopsin

  Terakita A., Koyanagi M., Tsukamoto H., Kubokawa K., Shichida Y.

  The Biophysical Society of Japan General Incorporated Association, 2005, Seibutsu Butsuri, 45, S101, Japanese


• 2P282 Comparative analyses of activation mechanisms of rhodopsin using site-directed mutagenesis

  Tsukamoto H., Terakita A., Shichida Y.

  The Biophysical Society of Japan General Incorporated Association, 2005, Seibutsu Butsuri, 45, S190, Japanese


• Counterion displacement in the molecular evolution of the rhodopsin family

  Akihisa Terakita, Mitsumasa Koyanagi, Hisao Tsukamoto, Takahiro Yamashita, Takashi Miyata, Yoshinori Shichida

  Mar. 2004, Nature Structural and Molecular Biology, 11(3) (3), 284 - 289, English

  [Refereed]

  Scientific journal


• 1P239 Comparative analyses of active state of varied rhodopsins by site-directed mutagenesis

  Terakita A., Tsukamoto H., Shichida Y.

  The Biophysical Society of Japan General Incorporated Association, 2004, Seibutsu Butsuri, 44, S89, Japanese


• 1P237 Analysis of retinal-opsin interaction in invertebrate-type rhodopsin having binding ability to agonist all-trans-retinal

  Tsukamoto H., Terakita A., Koyanagi M., Shichida Y.

  The Biophysical Society of Japan General Incorporated Association, 2004, Seibutsu Butsuri, 44, S89, Japanese


• Investigation of amino acid residues interacting with all-trans-retinal agonist in rhodopsin.

  Tsukamoto H., Terakita A., Koyanagi M., Miyata T., Shichida Y.

  The Biophysical Society of Japan General Incorporated Association, 2003, Seibutsu Butsuri, 43, S185, Japanese

• Characterization and engineering of a blue-sensitive, Gi/o-biased, and bistable ciliary opsin from a fan worm

  Sachiko Fukuzawa, Tomoki Kawaguchi, Takushi Shimomura, Yoshihiro Kubo, Hisao Tsukamoto

  Abstract Ciliary opsins have been identified not only in vertebrates but also in invertebrates. An invertebrate ciliary opsin was recently identified in the fan wormAcromegalomma interruptum(formerly namedMegalomma interrupta); however, its spectral and signaling characteristics are unknown. In the present study, we characterized the spectral properties and light-induced cellular signaling properties of the opsin (AcrInvC-opsin).AcrInvC-opsin showed an absorption maximum at 464 nm and upon blue-light absorption, the spectrum was red-shifted by approximately 50 nm. The two states are inter-convertible by illumination with blue and orange light. Blue light illumination ofAcrInvC-opsin caused specific coupling with Gi, sustained Gi dissociation, decreased intracellular cAMP levels, and activation of GIRK channels. The cellular responses by the activated opsin were partially terminated by orange light illumination. These light-dependent responses indicate that the InvC-opsin is a typical bistable pigment wherein the resting and activated states can be inter-converted by visible light illumination. We also attempted to modulate the spectral and functional properties ofAcrInvC-opsin using site-directed mutagenesis. Substitution of Ser-94 with Ala caused little spectral shift in the resting state but a further red-shift of ∼10 nm in the activated state, indicating that the absorption spectra of the two states were tuned differently. In contrast, the S94A substitution did not significantly affect the light-dependent signaling properties ofAcrInvC-opsin. BecauseAcrInvC-opsin is a blue-sensitive, Gi/o-biased, and bistable pigment, it has the potential to serve as an optical control tool to specifically and reversibly regulate Gi/o-dependent signaling pathways by visible light.

  Last, Cold Spring Harbor Laboratory, 21 Nov. 2024, bioRxiv, English

  Technical report


• Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

  Kohei Obayashi, Ruisi Zou, Toshifumi Mori, Hisao Tsukamoto

  Abstract Melanopsin functions in intrinsically photosensitive retinal ganglion cells of mammals to regulate circadian clock and pupil constriction. The opsinamide AA92593 has been reported to specifically inhibit mouse and human melanopsin functions as a competitive antagonist against retinal; however, the molecular mechanisms underlying its specificity have not been resolved. In this study, we attempted to identify amino acid residues responsible for the specific interaction of AA92593 with mammalian melanopsins. Our cell-based assays confirmed that AA92593 effectively inhibited the light-induced cellular responses of mammalian melanopsins, but not those of non-mammalian vertebrate and invertebrate melanopsins. These results suggest that amino acid residues specifically conserved among mammalian melanopsins are important for the antagonistic effect of AA92593, and we noticed Phe-94, Ser-188, and Ser-269 as candidate residues. Substitutions of these residues reduced the antagonistic effect of AA92593. We conducted docking and molecular dynamics simulations based on the AlphaFold-predicted melanopsin structure. The simulations indicated that Phe-94, Ser-188, and Ser-269 are located at the AA92593-binding site, and additionally identified Trp-189 and Leu-207 interacting with the antagonist. Substitutions of Trp-189 and Leu-207 affected the antagonistic effect of AA92593. Furthermore, substitutions of these amino acid residues converted AA92593-insensitive melanopsins susceptible to the antagonist. Based on experiments and molecular simulations, five amino acid residues, at positions 94, 188, 189, 207, and 269, were found to be responsible for the specific interaction with AA92593 in mammalian melanopsins.

  Last, Cold Spring Harbor Laboratory, 01 Nov. 2024, bioRxiv, English

  Technical report


• Development of Gβγ-Biased Optical Control Tools Based on an Invertebrate Opsin

  Hisao TSUKAMOTO

  Lead, Biophysical Society of Japan, Mar. 2024, Seibutsu Butsuri, 64(1) (1), 32 - 34, Japanese

  [Refereed][Invited]

  Introduction scientific journal


• Development of a “finely-biased” optical control tool using a self- inactivating photosensitive GPCR

  Hisao Tsukamoto

  Lead, Feb. 2024, SEIKAGAKU, 96(1) (1), 70 - 74, Japanese

  [Refereed][Invited]

  Introduction scientific journal


• A self-inactivating invertebrate opsin with resistance to retinal depletion optically drives biased signaling toward Gβγ-dependent ion channel modulation

  Hisao Tsukamoto, Yoshihiro Kubo

  Abstract Animal opsins, light-sensitive G protein-coupled receptors (GPCRs), have been utilized for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Ga and Gβγ subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Ga-, Gβγ-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Ga and Gβγ. Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin,Platynereisc-opsin1, drives biased signaling for Gβγ-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between Gβγ-dependent and Ga-dependent responses. The opsin-induced transient Gi/o activation preferably causes activation of the kinetically-fast Gβγ-dependent GIRK channels rather than slower Gi/oα-dependent adenylyl cyclase inhibition. Although similar Gβγ-biased signaling properties were observed in a selfinactivating vertebrate visual pigment,Platynereisc-opsin1 needs fewer retinal molecules to evoke cellular responses. Furthermore, the Gβγ-biased signaling properties ofPlatynereisc-opsinl are enhanced by genetically fused with RGS8 protein which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for Gβγ-dependent ion channel modulation.

  Lead, Cold Spring Harbor Laboratory, 06 Jan. 2023, bioRxiv, English

  Technical report


• Functional characteristics of animal opsins as optical control tools.

  Hisao TSUKAMOTO

  Lead, The Japanese Society for Comparative Physiology and Biochemistry, 01 Aug. 2022, Hikaku seiri seikagaku(Comparative Physiology and Biochemistry), 39(2) (2), 84 - 91, Japanese

  [Refereed][Invited]

  Introduction scientific journal


• Optical control of cellular signaling pathways using animal opsins

  Hisao Tsukamoto

  Lead, SPIE, 27 Oct. 2021, Biomedical Imaging and Sensing Conference 2021, 119250E, English

  Introduction international proceedings

  Link to Kobe Univ. Repository (Kernel)


• オーファン助教の延長戦

  塚本寿夫

  分子科学研究所, Mar. 2021, 分子研レターズ, 83, 26 - 27, Japanese


• 【書評】細胞のマルチスケールメカノバイオロジー

  塚本 寿夫

  一般社団法人 日本生物物理学会, 2017, 生物物理, 57(5) (5), 278 - 278, Japanese


• 出前授業 生き物の「分子」と「進化」

  自然科学研究機構, Sep. 2015, 自然科学研究機構 広報誌OKAZAKI, 48, Japanese, Domestic magazine


• Conformational Dynamics of a G Protein-Coupled Receptor Opsin and Its Constitutively Active Mutant

  TSUKAMOTO Hisao

  The Biophysical Society of Japan General Incorporated Association, 2014, Seibutsu Butsuri, 54(2) (2), 111 - 112, Japanese


• Arrestin can Bind to a Single G-Protein Coupled Receptor

  Hisao Tsukamoto, Abhinav Sinha, Mark DeWitt, David L. Farrens

  Jan. 2010, BIOPHYSICAL JOURNAL, 98(3) (3), 291A - 291A, English

  Summary international conference


• Report of 4th Asia Oceania Conference on Photobiology (AOCP)

  Hisao TSUKAMOTO

  Lead, Biophysical Society of Japan, 2009, Seibutsu Butsuri, 49(2) (2), 098 - 099, Japanese

  [Refereed][Invited]

  Meeting report


• Comparative biochemical analysis of invertebrate visual pigments and melanopsin

  Akihisa Terakita, Hisao Tsukamoto, Mitsumasa Koyanagi, Michio Sugahara, Takahiro Yamashita, Yoshinori Shichida

  Nov. 2007, COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 148(3) (3), 345 - 345, English

  Summary international conference


• 【五感の科学】 視覚を担うGタンパク質共役型受容体ロドプシン

  塚本 寿夫, 寺北 明久, 七田 芳則

  (公社)日本薬学会, May 2007, ファルマシア, 43(5) (5), 432 - 436, Japanese


• Comparative analyses of melanopsin with invertebrate visual pigmetns

  Akihisa Terakita, Mitsumasa Koyanagi, Hisao Tsukamoto, Yoshinori Shichida

  Dec. 2006, ZOOLOGICAL SCIENCE, 23(12) (12), 1198 - 1198, English

  Summary international conference


• Diversity of the vertebrate non-visual pigment parapinopsins

  Mitsumasa Koyanagi, Akihisa Terakita, Hisao Tsukamoto, Akihisa Terakita

  Dec. 2006, ZOOLOGICAL SCIENCE, 23(12) (12), 1198 - 1198, English

  Summary international conference


• ロドプシン類の分子進化

  塚本 寿夫, 寺北 明久, 七田 芳則

  (公財)金原一郎記念医学医療振興財団, Oct. 2006, 生体の科学, 57(5) (5), 500 - 501, Japanese


• A rhodopsin exhibiting binding ability to agonist all-trans-retinal

  Hisao Tsukamoto, Akihisa Terakita, Yoshinori Shichida

  Dec. 2005, ZOOLOGICAL SCIENCE, 22(12) (12), 1478 - 1479, English

  Summary international conference


• EXPRESSION AND CHARACTERIZATION OF LANCELET OPSIN(Physiology)(Proceeding of the Seventy-Third Annual Meeting of the Zoological Society of Japan) :

  Tsukamoto H., Terakita A., Koyanagi M., Miyata T., Shichida Y.

  Zoological Society of Japan, 2001, Zoological science, 18, 110 - 110, English

• 光と生命の事典

  塚本 寿夫

  Contributor, 無脊椎動物オプシン, 朝倉書店, 2016


• The evolution of visual and non-visual pigments

  Hisao Tsukamoto

  Contributor, Diversity and functional properties of bistable photopigments, Springer, 2014


• 動物の多様な生き方１ 見える光，見えない光

  寺北明久, 塚本寿夫

  Contributor, 光を受容するさまざまな分子, 共立出版, 2009

• 動物の光受容タンパク質オプシンの歴史・特徴・応用

  塚本 寿夫

  東京大学物性研究所機能物性セミナー, Feb. 2025, Japanese

  [Invited]


• Biochemical and computational analyses underlying the specific recognition of mammalian melanopsins with a small molecule AA92593

  尾林虹兵, 鄒瑞思, 森俊文, 塚本寿夫

  日本動物学会 第95回長崎大会, Sep. 2024, Japanese

  Oral presentation


• 多様なGi/o共役型オプシンの活性化と不活性化に伴う、シグナル伝達分子の動態の解析

  松本怜之, 成宮毱那, 塚本寿夫

  日本動物学会 第95回長崎大会, Sep. 2024, Japanese

  Oral presentation


• 種々の動物種が持つメラノプシンのGタンパク質共役特異性の違いとその制御メカニズムの解析

  佐竹 うらら, 尾林 虹兵, 塚本 寿夫

  日本動物学会 第95回長崎大会, Sep. 2024, Japanese

  Oral presentation


• Characterization of an invertebrate opsin having bi-stable and bi-coupling properties.

  川口倫輝, 佐藤恵太, 大内淑代, 塚本寿夫

  日本動物学会 第95回長崎大会, Sep. 2024, Japanese

  Oral presentation


• Optical modulation of ion channel activity by opsin, light-sensitive GPCR

  Hisao Tsukamoto

  Ion Channel Modulation Symposium - Japan 2024, May 2024, English

  [Invited]

  Invited oral presentation


• Molecular characteristics of an invertebrate Gi/o-coupled and visible light-sensitive opsin

  Sachiko Fukuzawa, Hisao Tsukamoto

  第61回日本生物物理学会年会, Nov. 2023, English

  Poster presentation


• Development of a Gs-coupled optogenetic tool that can be turned on and off by visible light

  Akinari Sakayori, Yusuke Sakai, Naoyuki Taira, Yusei Sakata, Mitsumasa Koyanagi, Akihisa Terakita, Saori Tani-Matsuhana, Kunio Inoue, Hisao Tsukamoto

  第61回日本生物物理学会年会, Nov. 2023, English

  Poster presentation


• Analysis of invertebrate-type opsin-mediated G protein activation and downstream signaling kinetics using luminescent biosensors

  Marina Narimiya, Michihiro Ohta, Hisao Tsukamoto

  第61回日本生物物理学会年会, Nov. 2023, English

  Poster presentation


• Biochemical and computational analyses of interactions between mammalian melanopsins and a specific antagonist

  Kohei Obayashi, Ruisi Zou, Toshifumi Mori, Hisao Tsukamoto

  第61回日本生物物理学会年会, Nov. 2023, English

  Poster presentation


• Characterization of an opsin having bi-stable and bi-coupling properties

  Tomoki Kawaguchi, Hisao Tsukamoto

  第61回日本生物物理学会年会, Nov. 2023, English

  Poster presentation


• 動物オプシンの分子特性とその機能チューニング

  塚本寿夫

  第３回神戸大学次世代光散乱イメージング科学研究センターシンポジウム, Sep. 2023, Japanese

  [Invited]

  Invited oral presentation


• 非視覚の光受容に関わるオプシンを光操作ツールとして活用する

  塚本寿夫

  日本動物学会 第94回山形大会, Sep. 2023, Japanese

  [Invited]

  Invited oral presentation


• 無脊椎動物オプシンを「よい」 光操作ツールに改変する

  塚本寿夫

  生理研研究会 「構造情報を基盤とした膜機能分子の生理機能理解に向けて」, Sep. 2023, Japanese

  [Invited]

  Invited oral presentation


• 無脊椎動物オプシンの特性を生かしたGβγシグナル経路選択的光操作ツールの開発

  塚本寿夫

  第2回 神戸大学次世代光散乱イメージング科学研究センターシンポジウム, Jan. 2023, Japanese

  [Invited]

  Invited oral presentation


• Characterization of an invertebrate opsin having transient G protein-activating state(s) as optical control tool

  Hisao Tsukamoto, Yoshihiro Kubo

  THE 19TH INTERNATIONAL CONFERENCE ON RETINAL PROTEINS, Oct. 2022, English

  Poster presentation


• Development of bistable optical control tools based on a Gs-coupled opsin

  Akinari Sakayori, Yusuke Sakai, Mitsumasa Koyanagi, Akihisa Terakita, Hisao Tsukamoto

  第60回日本生物物理学会年会, Sep. 2022, English

  Poster presentation


• An invertebrate opsin functionally biased for Gbg-dependent ion channel responses

  Hisao Tsukamoto, Yoshihiro Kubo

  第60回日本生物物理学会年会, Sep. 2022, English

  Poster presentation


• NA

  Hisao Tsukamoto, Yoshihiro Kubo

  第22回日本光生物学協会年会, Aug. 2022, Japanese

  Poster presentation


• 動物の光受容タンパク質オプシンの多様性を光操作ツールの視点から見る

  塚本寿夫

  自然科学研究機構 「ネットワーク型研究加速事業（国際ネットワーク）」生理研プロジェクト 最終成果報告会, Mar. 2022, Japanese

  [Invited]

  Invited oral presentation


• Functional diversity of animal opsins as optical control tools

  塚本寿夫

  レーザー学会学術講演会第42回年次大会, Jan. 2022, Japanese

  [Invited]

  Invited oral presentation


• 光操作ツールとして無脊椎動物オプシンが持つ利点

  塚本寿夫

  CSMIワークショップ Emergence Conference, Dec. 2021, Japanese

  [Invited]

  Invited oral presentation


• リサイクル型オプシンの光応答特性を利用した光操作ツールの開発

  塚本寿夫

  生理研研究会「構造情報を基盤とした膜機能分子の生理機能理解に向けて」, Sep. 2021

  [Invited]

  Invited oral presentation


• Optical control of cellular responses using invertebrate opsin

  Hisao Tsukamoto, Yoshihiro Kubo

  日本動物学会第92回オンライン米子大会, Sep. 2021

  Poster presentation


• Optical control of cellular signaling pathways using animal opsins

  Hisao Tsukamoto

  OPTICS & PHOTONICS International Congress 2021, Apr. 2021, English

  [Invited]

  Invited oral presentation


• 無脊椎動物オプシンの光遺伝学ツールとしての可能性

  塚本 寿夫

  第4回極みプロジェクトシンポジウム, Sep. 2020, Japanese

  [Invited]

  Invited oral presentation


• Biological researches I had conducted at IMS

  Hisao Tsukamoto

  分子研コロキウム, Jul. 2020, Japanese

  [Invited]

  Public discourse


• ”総力戦”としての光操作技術

  塚本寿夫

  第 57 回日本生物物理学会年会, Sep. 2019, English


• 無脊椎動物の繊毛型オプシンの多様なシグナル伝達特性とそれを利用した光操作ツールの作製

  塚本寿夫

  日本動物学会第 90 回大阪大会, Sep. 2019, Japanese

  Oral presentation


• 無脊椎動物オプシンの物性を利用してイオンチャネルを光操作する

  塚本寿夫

  ISSP ワークショップ「レチナールタンパク質の光機能発現の物理と化学」, Sep. 2019, Japanese


• チャネル機能の操作ツールとしての無脊椎動物光受容体

  塚本寿夫

  生理研研究会「イオンチャネルと生体膜のダイナミズム:構造生物学の先にあるもの」, Sep. 2019, Japanese


• Investigation of ligand-protein interaction in a G protein-coupled receptor via ATR-FTIR spectroscopy.

  H. Tsukamoto, Y. Furutani

  第56回日本生物物理学会年会, Sep. 2018, Japanese

  Poster presentation


• 無脊椎動物が持つ繊毛型オプシンが示す多様な機能特性

  塚本寿夫, 古谷祐詞

  第 20 回日本光生物学協会年会, Aug. 2018, Japanese

  Poster presentation


• 動物プランクトンの脳内紫外光受容体が持つ分子特性

  塚本寿夫, I-Shan Chen, 久保義弘, 古谷祐詞

  生理学研究所プロジェクト「機能タンパク質の構造と機能のダイナミクスと、それに基づく細胞・ 生体システム作動機構の研究拠点の形成」平成 29 年度末シンポジウム, Mar. 2018, Japanese


• Functional properties and the regulating mechanisms of a mammalian two-pore domain potassium channel TWIK-1.

  H. Tsukamoto, K. Nakajo, Y. Kubo, Y. Furutani

  第55回日本生物物理学会年会, Sep. 2017, Japanese

  Poster presentation


• 動物プランクトンの脳ではたらく繊毛型オプシンの分子特性

  塚本寿夫, I-Shan Chen, 久保義弘, 古谷祐詞

  日本動物学会第88回富山大会, Sep. 2017, Japanese


• 動物プランクトンの脳ではたらく紫外光受容体の分光・電気生理解析

  塚本寿夫, I-Shan Chen, 久保義弘, 古谷祐詞

  第2回イオンチャネル研究会, Aug. 2017, Japanese


• Spectral and biochemical characterization of mammalian melanopsins (Opn4s) and invertebrate Opn3 homologs.

  H. Tsukamoto, Y. Kubo, I. S. Chen, D. L. Farrens, M. Koyanagi, A. Terakita, Y. Furutani

  17th International Conference on Retinal Proteins, Oct. 2016, English


• 哺乳類カリウムチャネル TWIK-1 の機能特性と、赤外分光を用いたイオンとの相互作用の解析

  塚本寿夫, 中條浩一, 久保義弘, 古谷祐詞

  平成28年度 生理研研究会 「膜システムの機能的・構造的統合」, Sep. 2016, Japanese


• 無脊椎動物における繊毛型オプシンの分子特性と非視覚の光受容

  塚本寿夫

  第 19 回日本光生物学協会年会, Jul. 2016, Japanese


• 哺乳類における「非視覚」の光受容機能を支えるメラノプシンの分子基盤

  塚本寿夫, 久保義弘, David Farrens, 小柳光正, 寺北明久, 古谷祐詞

  第 43 回 生体分子科学討論会, Jun. 2016, Japanese

  Oral presentation


• 哺乳類メラノプシンにおける、レチナール結合の「揺らぎ」

  塚本寿夫, 久保義弘, David Farrens, 小柳光正, 寺北明久, 古谷祐詞

  自然科学研究機構 3 プロジェクト終了シンポジウム「次ステージ機能生命科学の展望」, Mar. 2016, Japanese


• Molecular mechanisms regulating function of photoreceptive proteins

  Hisao Tsukamoto

  JSPS Summer Program 2015, Jun. 2015, English


• Energetics and conformational dynamics underlying the activation of the G protein-coupled receptor opsin assessed by site-directed fluorescence labeling and nanodisc techniques.

  H. Tsukamoto, D. L. Farrens

  6th International Conference on Retinal Proteins, Oct. 2014, English

  Oral presentation


• 哺乳動物の概日リズムの調節を担う光受容タンパク質メラノプシンの分子特性

  塚本寿夫, David Farrens, 久保義弘, 小柳光正, 寺北明久, 古谷祐詞

  日本動物学会第83回大会, Aug. 2012, Japanese

  Oral presentation


• 光受容タンパク質ロドプシンの機能調節メカニズム

  塚本寿夫

  2011年度第3回名古屋支部セミナー 「生化名古屋・夏のセミナー」, Aug. 2011, Japanese

  [Invited]

• The Biophysical Society of Japan

  Sep. 2003 - Present


• The Zoological Society of Japan

  Sep. 2001 - Present

• AQP4-dependent ATP/Adenosine releaser from astrocyte following neuronal activities

  森田 光洋, 塚本 寿夫

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Kobe University, 01 Apr. 2022 - 31 Mar. 2025


• Construction of further biased optical control tools manipulating GPCR signalings

  塚本 寿夫, 森田 光洋

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Kobe University, 01 Apr. 2021 - 31 Mar. 2025


• 内在受容体を利用した生命機能の新規光操作手法の開発

  塚本 寿夫

  科学技術振興機構, 戦略的創造研究推進事業 さきがけ, 分子科学研究所、神戸大学, Oct. 2017 - Sep. 2021, Principal investigator

  この十数年間に、外来の(異なる生物種由来の)光受容タンパク質遺伝子を強制発現させて、動物の神経活動などを光で操作する技術は爆発的に発展しています。この研究提案では、外来遺伝子を用いるのではなく、研究対象の動物に内在する受容体遺伝子を活用して生命機能を光操作する技術の開発に取り組みます。そして、これまで以上に幅広い動物種・生命機能に光操作技術を適用できるようにします。


• Investigation of molecular mechanisms underlying environment-dependent regulation of ion permeation through mammalian potassium channels.

  Tsukamoto Hisao

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists (B), Institute for Molecular Science, 01 Apr. 2017 - 31 Mar. 2020

  Generally, potassium ion channels selectively permeate potassium ions and block sodium ions to generate potentials across biological membranes. However, some potassium channels, such as a two-pore domain potassium channel TWIK-1, can permeate sodium ions under specific conditions. It is poorly understood how the potassium channels regulate their ion selectivity. We aimed to reveal molecular mechanisms underlying the "loose" ion selectivity of TWIK-1 using infrared and fluorescence spectroscopic techniques. Infrared spectroscopy revealed that the selective filter moiety in TWIK1 possesses lower the affinity for potassium ions. Also, fluorescence spectroscopy showed that an "entry" site for ions in the extracellular domain of TWIK-1 becomes more opened depending on sodium concentrations. These results suggested that the selectivity filter and the entry site in the extracellular domain cooperatively play important roles in unconventional sodium permeability in TWIK-1.


• Analyses of molecular characteristics in mammalian melanopsins to receive ambient light signals.

  TSUKAMOTO Hisao

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists (B), Institute for Molecular Science, 01 Apr. 2013 - 31 Mar. 2015

  As other animals, mammals receive ambient light signals to regulate their circadian clocks. Such a "non-visual" photoreception in mammals is mediated by not only visual photoreceptor cells but also intrinsically retinal ganglion cells. A photoreceptive protein named melanopsin receives and transmits the light signals in the intrinsically retinal ganglion cells. In this study, we compared biochemical and spectroscopic properties of mammalian melanopsins with those of closely related photopigments with an aim to identify some molecular characteristics in mammalian melanopsins suited for the non-visual photoreception. Our data suggested that mammalian melanopsins have acquired some characteristics to receive ambient light signals.


• Functional expression of mammalian ion channels and analysis of the molecular mechanisms

  FURUTANI Yuji, NAKAJO Koichi, TSUKAMOTO Hisao, KIMURA Tetsunari, KUBO Yoshihiro

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Exploratory Research, Institute for Molecular Science, 01 Apr. 2012 - 31 Mar. 2014

  Ion channel proteins work for selective permeation of ions in response to various stimuli and regulate electrical signals in living organisms. To understand the molecular mechanisms of these channels, the precise structural information with atomic resolution is required. In this study, mammalian ion channel proteins, which are important targets in medical and pharmaceutical sciences, have been studied by attenuated total reflection (ATR) FT-IR spectroscopy which gives us precise molecular information. After screening of several ion channel proteins, we have succeeded to resolve amide I bands of selectivity filter of KCNK1 channel, which is involved in regulation of resting membrane potential of cell, by using ATR FT-IR spectroscopy.


• 体内時計の調節を担う光受容体メラプシンの機能発現メカニズムの解析

  塚本寿夫

  日本学術振興会, 海外特別研究員, Oregon Health & Science University, Apr. 2011 - Jun. 2011, Principal investigator


• G蛋白質共役受容体の多量体化の機能的意義

  塚本寿夫

  上原記念生命科学財団, リサーチフェローシップ, Oregon Health & Science University, Apr. 2010 - Mar. 2011


• 光受容蛋白質の機能多様性とそれを生み出す分子特性の分子生理学・生化学的解析

  塚本 寿夫

  日本学術振興会, 科学研究費助成事業, 特別研究員奨励費, 大阪市立大学, Apr. 2007 - Mar. 2010

  本研究課題では、動物の視覚などを担う「普通」のロドプシンおよびその類似タンパク質(以下ロドプシン類と呼ぶ)とは正反対の全トランス型から11シス型への発色団レチナールの異性化反応を起こすロドプシン類であるペロプシンやレチノクロムが機能発現する際に必要な分子メカニズムが、「普通」のロドプシンとどのように異なるのか(似ているのか)を明らかにすることを目指して、研究を行っている。 前年度までの研究から、通常のロドプシン類を調製する条件(界面活性剤で可溶化)では、ペロプシン・レチノクロムの性質が大きく変化してしまうことが明らかになっていた。また前年度までにグリセリンを添加することにより界面活性剤の影響を緩和できることもわかっていた。そこで今年度は、主にレチノクロムを用いて、グリセリンを添加した試料あるいは、脂質存在下で界面活性剤を除去し、脂質小胞(リポソーム)に再構成した試料を用いて、機能発現メカニズムを解析した。 「普通」のロドプシン類を用いた実験では、6番目の膜貫通ヘリックスの特定の部位に蛍光プローブを導入し、その蛍光特性を光受容(レチナールの光異性化)前後で測定することにより、それらの機能発現メカニズムを解析することができる。そこで、レチノクロムについて6番目の膜貫通ヘリックスの同じ位置に蛍光プローブを導入したタンパク質を調製し、それらの蛍光特性をグリセリン添加条件、リポソーム再構成条件の2つの条件で測定した。その結果、レチノクロムでは「普通」のロドプシン類が光を受容する際に見られるような蛍光特性の変化が見られなかった。この結果は、少なくともレチノクロムが光を受容する際には、「普通」のロドプシン類とは異なるメカニズムで機能が発現することを示唆している。


• 変異蛋白質を用いたナメクジウオロドプシンと多様な視物質との性質・機能の比較解析

  塚本 寿夫

  日本学術振興会, 科学研究費助成事業, 特別研究員奨励費, 京都大学, Apr. 2003 - Mar. 2006

  ロドプシン類似の光受容蛋自質は、アミノ酸配列の相同性に基づき5種類のサブグループに分類できる。本研究は、ロドプシン類における活性化機構の類似点と相違点を明らかにすることを目的とする。具体的には、脊椎動物の視物質とは異なるサブグループに属するナメクジウオロドプシンの機能発現機構を、変異蛋白質を用いて解析し、よく研究されている脊椎動物の視物質(ウシロドプシン)と比較した。前年度までの研究から、ナメクジウオロドプシンの6番目の膜貫通ヘリックスに存在するAla269残基に変異を導入すると、光産物(活性状態)の吸収スペクトルが大きく変化し、G蛋白質活性化能が低下することを見いだした。なぜAla269変異が光産物に大きく影響するのか明らかにすることはナメクジウオロドプシンの機能発現機構の理解につながると考え、以下の実験を行った。 1.Ala269変異の影響を小さくするAla269周辺の残基の変異を探索した。その結果5番目の膜貫通ヘリックスに存在するアミノ酸残基に変異を導入すると、Ala269変異の影響が小さくなった。一方、ウシロドプシンのAla269に変異を導入するとG蛋白質活性化能の低下は見られたが、光産物の吸収スペクトルへの影響はナメクジウオロドプシンの場合と異なった。 2.ウシロドプシンにおいては、活性状態への構造変化(構成的活性化)を誘起する変異が知られている。その中の代表的な変異をナメクジウオロドプシンのAla269変異体において対応する位置に導入した。その結果光産物の吸収スペクトルが野生型様に戻ることが示唆された。 以上の結果から、ナメクジウオロドプシンが活性状態を形成する上で5番目と6番目の膜貫通ヘリックスのAla269周辺領域が構造変化することを見いだした。ウシロドプシンにおいても同様の構造変化は重要であるが、ナメクジウオロドプシンとは相違点も存在する可能性を見いだした。