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Light-dark Adaptation of Channelrhodopsin C128T Mutant*

  • Eglof Ritter
    Correspondence
    To whom the correspondence may be addressed: Institut für Medizinische Physik und Biophysik, Charité, Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Tel.: 49-30-450524196; Fax: 49-30-450-524-952;
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Patrick Piwowarski
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Peter Hegemann
    Affiliations
    Institut für Biologie, Humboldt-Universität zu Berlin, Unter den Linden 6, 10099 Berlin, Germany
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  • Franz J. Bartl
    Correspondence
    To whom the correspondence may be addressed: Institut für Medizinische Physik und Biophysik, Charité, Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Tel.: 49-30-450524196; Fax: 49-30-450-524-952;
    Affiliations
    Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany
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  • Author Footnotes
    * This work was supported by the Deutsche Forschungsgemeinschaft Grants BA 2242/2-1 (to F. J. B.) and SFB 1078.
    This article contains supplemental Figs. 1 and 2.
Open AccessPublished:February 25, 2013DOI:https://doi.org/10.1074/jbc.M112.446427
      Channelrhodopsins are microbial type rhodopsins that operate as light-gated ion channels. Largely prolonged lifetimes of the conducting state of channelrhodopsin-2 may be achieved by mutations of crucial single amino acids, i.e. cysteine 128. Such mutants are of great scientific interest in the field of neurophysiology because they allow neurons to be switched on and off on demand (step function rhodopsins). Due to their slow photocycle, structural alterations of these proteins can be studied by vibrational spectroscopy in more detail than possible with wild type. Here, we present spectroscopic evidence that the photocycle of the C128T mutant involves three different dark-adapted states that are populated according to the wavelength and duration of the preceding illumination. Our results suggest an important role of multiphoton reactions and the previously described side reaction for dark state regeneration. Structural changes that cause formation and depletion of the assumed ion conducting state P520 are only small and follow larger changes that occur early and late in the photocycle, respectively. They require only minor structural rearrangements of amino acids near the retinal binding pocket and are triggered by all-trans/13-cis retinal isomerization, although additional isomerizations are also involved in the photocycle. We will discuss an extended photocycle model of this mutant on the basis of spectroscopic and electrophysiological data.

      Introduction

      Channelrhodopsin (ChR)
      The abbreviations used are: ChR
      channelrhodopsin
      IDA
      initial dark state
      DAB
      dark-adapted state after blue
      DAG
      dark-adapted state after green illumination.
      is a microbial type rhodopsin that serves as a light-gated ion channel in the eye spot of green algae. As typical for rhodopsins, it exhibits seven transmembrane helices. Similar to bacteriorhodopsin and other microbial rhodopsins, it undergoes a photocycle initiated by light-induced isomerization of the retinal chromophore. Details of the molecular architecture of its dark-adapted state are now available owing to the crystal structure of a chimera between ChR1 and ChR2 (
      • Kato H.E.
      • Zhang F.
      • Yizhar O.
      • Ramakrishnan C.
      • Nishizawa T.
      • Hirata K.
      • Ito J.
      • Aita Y.
      • Tsukazaki T.
      • Hayashi S.
      • Hegemann P.
      • Maturana A.D.
      • Ishitani R.
      • Deisseroth K.
      • Nureki O.
      Crystal structure of the channelrhodopsin light-gated cation channel.
      ). This structure allows the identification of residues in the retinal binding pocket and the putative cation-conducting pathway.
      However, molecular changes that are linked to formation of the conducting pore and stabilization of the channel are not yet understood. Electrical measurements yielded the first information about the channelrhodopsin photocycle. On and off kinetics of the photocurrents has provided insights into the formation and decay of the conducting state in the microsecond time range (
      • Bamann C.
      • Kirsch T.
      • Nagel G.
      • Bamberg E.
      Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function.
      ,
      • Ernst O.P.
      • Sánchez Murcia P.A.
      • Daldrop P.
      • Tsunoda S.P.
      • Kateriya S.
      • Hegemann P.
      Photoactivation of channelrhodopsin.
      ).
      UV-visible absorption measurements on recombinant ChR identified two early photocycle intermediates, P500 and P380, that precede the conducting state P520 (
      • Bamann C.
      • Kirsch T.
      • Nagel G.
      • Bamberg E.
      Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function.
      ,
      • Ernst O.P.
      • Sánchez Murcia P.A.
      • Daldrop P.
      • Tsunoda S.P.
      • Kateriya S.
      • Hegemann P.
      Photoactivation of channelrhodopsin.
      ). The latter decays within 10 ms, but complete regeneration of the dark state is slow and occurs in the time range of 20 s (
      • Ritter E.
      • Stehfest K.
      • Berndt A.
      • Hegemann P.
      • Bartl F.J.
      Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.
      ,
      • Nagel G.
      • Szellas T.
      • Huhn W.
      • Kateriya S.
      • Adeishvili N.
      • Berthold P.
      • Ollig D.
      • Hegemann P.
      • Bamberg E.
      Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.
      ). The late photocycle intermediate P480 is photochemically active; the photocurrents decline to a reduced stationary level upon repetitive or prolonged ChR2 stimulation. One solution for the interpretation of this observation comes from the ChR2 mutants called step function rhodopsin. In these mutants, Cys-128 is exchanged by threonine, serine, or alanine, thereby dramatically slowing down the lifetime of the conducting state. Additionally, these mutants can be switched on and off by blue and green light, respectively. These properties offer the opportunity to achieve a more permanent cell depolarization, where the lifetime of the open state can be controlled by flash or continuous low light intensities (
      • Berndt A.
      • Yizhar O.
      • Gunaydin L.A.
      • Hegemann P.
      • Deisseroth K.
      Bi-stable neural state switches.
      ).
      The photocycle of such mutants is however more complex than originally anticipated for the ChR2 wild type. It exhibits branches and side reactions that render step function rhodopsins nonfunctional after extended illumination (
      • Zhang Y.P.
      • Oertner T.G.
      Optical induction of synaptic plasticity using a light-sensitive channel.
      ,
      • Stehfest K.
      • Ritter E.
      • Berndt A.
      • Bartl F.
      • Hegemann P.
      The branched photocycle of the slow-cycling channelrhodopsin-2 mutant C128T.
      ,
      • Schoenenberger P.
      • Schärer Y.P.
      • Oertner T.G.
      Channelrhodopsin as a tool to investigate synaptic transmission and plasticity.
      ). We previously suggested that the probability of photochemical side reactions increases with the lifetime of the corresponding state (
      • Stehfest K.
      • Ritter E.
      • Berndt A.
      • Bartl F.
      • Hegemann P.
      The branched photocycle of the slow-cycling channelrhodopsin-2 mutant C128T.
      ,
      • Stehfest K.
      • Hegemann P.
      Evolution of the channelrhodopsin photocycle model.
      ). Despite these complications the slow kinetics of step function rhodopsins offers the opportunity to study the photocycle of Cys-128 in depth because they facilitate the application of FTIR spectroscopy, which allows the observation of the reaction mechanisms of channelrhodopsins at the molecular level.
      In this study, we reinvestigated the photocycle of the slow cycling mutant C128T by a combination of FTIR and UV-visible spectroscopy and by retinal extraction with subsequent HPLC analysis. We show the existence of two dark intermediates that are populated depending on the wavelength and duration of the preceding illumination. Formation of the presumed ion conducting state P520 (channel gating) involves two distinct mechanistic steps. Formation and decay of a pre-conducting state occurs early and late in the photocycle and requires major backbone alterations. Second, a fast switch between a pre-conducting state and P520, triggered by all-trans/13-cis and/or syn/anti Schiff base isomerization requires only minor structural changes in or near the binding pocket. Finally, we present a model of the photocycle that links and explains both spectroscopic and electrophysiological findings.

      Acknowledgments

      We acknowledge Brian Bauer, Anja Koch, and Roman Kazmin for technical support in sample preparation and Martha Sommer for critical reading of the manuscript.

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