title: Chimeras of channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii exhibit distinctive light-induced structural changes from channelrhodopsin-2.
abstract: Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.
authors: From the Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan, PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, Department of Structural Molecular Science, Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.
authors: Asumi
authors: A
authors: Inaguma
authors: From the Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan, Department of Structural Molecular Science, Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.
authors: Hisao
authors: H
authors: Tsukamoto
authors: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors: Hideaki E
authors: HE
authors: Kato
authors: From the Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan, Department of Structural Molecular Science, Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.
authors: Tetsunari
authors: T
authors: Kimura
authors: CREST, Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, and Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai 980-8577, Japan.
authors: Toru
authors: T
authors: Ishizuka
authors: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors: Satomi
authors: S
authors: Oishi
authors: CREST, Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, and Department of Developmental Biology and Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai 980-8577, Japan.
authors: Hiromu
authors: H
authors: Yawo
authors: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
authors: Osamu
authors: O
authors: Nureki
authors: From the Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan, PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, Department of Structural Molecular Science, Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan, furutani@ims.ac.jp.
authors: Yuji
authors: Y
authors: Furutani
mesh_terms: D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii; D015640:Ion Channel Gating; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D012243:Rhodopsin
keywords: Channel Desensitization; Channelrhodopsin; Fourier Transform IR (FTIR); Gating; Ion Channel; Photobiology; Proton Transfer; Rhodopsin