Abstract
The light-gated cation channel Channelrhodopsin-2 (ChR2), a retinylideneprotein found in the eye-spot of Chlamydomonas reinhardtii, became an optogenetic tool to trigger neurophysiological responses by light and, thus, revolutionized spatio-temporal studies of such processes. The reaction mechanism still remains elusive but recent vibrational spectroscopic experiments started to resolve details of the associated structural changes during the photocycle. Large alterations in the polypeptide backbone were observed by FT-IR spectroscopy that precede and succeed the opening and closing of the channel, respectively. However, the molecular switch that controls gating is still unknown. Here, we present difference spectra of the D156E mutant of ChR2 and assign the observed vibrational bands to crucial hydrogen bonding changes of this residue in various intermediate states of the photoreaction. By comparison with spectra of wild-type ChR2 and the C128T mutant and correlation to electrophysiological studies, we propose the DC gate as a crucial hydrogen-bonding interaction between D156 and C128 which may represent the valve of the channel.
Similar content being viewed by others
Notes and references
K. W. Foster, J. Saranak, N. Patel, G. Zarilli, M. Okabe, T. Kline and K. Nakanishi, A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas, Nature, 1984, 311, 756.
G. Nagel, T. Szellas, W. Huhn, S. Kateriya, N. Adeishvili, P. Berthold, D. Ollig, P. Hegemann and E. Bamberg, Channelrhodopsin-2, a directly light-gated cation-selective membrane channel, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 13940.
O. A. Sineshchekov, K. H. Jung and J. L. Spudich, Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 8689.
R. H. Kramer, D. L. Fortin and D. Trauner, New photochemical tools for controlling neuronal activity, Curr. Opin. Neurobiol., 2009, 19, 544.
O. P. Ernst, P. A. Sanchez Murcia, P. Daldrop, S. P. Tsunoda, S. Kateriya and P. Hegemann, Photoactivation of channelrhodopsin, J. Biol. Chem., 2008, 283, 1637.
C. Bamann, T. Kirsch, G. Nagel and E. Bamberg, Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function, J. Mol. Biol., 2008, 375, 686.
I. Radu, C. Bamann, M. Nack, G. Nagel, E. Bamberg and J. Heberle, Conformational changes of channelrhodopsin-2, J. Am. Chem. Soc., 2009, 131, 7313.
E. Ritter, K. Stehfest, A. Berndt, P. Hegemann and F. J. Bartl, Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy, J. Biol. Chem., 2008, 283, 35033.
C. Kötting and K. Gerwert, Proteins in action monitored by time-resolved FTIR spectroscopy, ChemPhysChem, 2005, 6, 881.
I. Radu, M. Schleeger, C. Bolwien and J. Heberle, Time-Resolved FT-IR Difference Spectroscopy and the Application to Membrane Proteins, Photochem. Photobiol. Sci., 2009, 8, 1517.
W. Mäntele, Reaction-induced infrared difference spectroscopy for the study of protein function and reaction mechanisms, Trends Biochem. Sci., 1993, 18, 197.
A. Maeda, Application of FTIR spectroscopy to the structural study on the function of bacteriorhodopsin, Isr. J. Chem., 1995, 35, 387.
K. Edman, A. Royant, P. Nollert, C. A. Maxwell, E. Pebay-Peyroula, J. Navarro, R. Neutze and E. M. Landau, Early structural rearrangements in the photocycle of an integral membrane sensory receptor, Structure, 2002, 10, 473.
A. Barth, The infrared absorption of amino acid side chains, Prog. Biophys. Mol. Biol., 2000, 74, 141.
C. Bamann, R. Gueta, S. Kleinlogel, G. Nagel and E. Bamberg, Structural Guidance of the Photocycle of Channelrhodopsin-2 by an Interhelical Hydrogen Bond, Biochemistry, 2009 DOI: 10.1021/bi901634p.
G. Nagel, T. Szellas, S. Kateriya, N. Adeishvili, P. Hegemann and E. Bamberg, Channelrhodopsins: directly light-gated cation channels, Biochem. Soc. Trans., 2005, 33, 863.
M. Nack, I. Radu, C. Bamann, E. Bamberg and J. Heberle, The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy, FEBS Lett., 2009, 583, 3676.
B. Nie, J. Stutzman and A. Xie, A vibrational spectral maker for probing the hydrogen-bonding status of protonated Asp and Glu residues, Biophys. J., 2005, 88, 2833.
Author information
Authors and Affiliations
Corresponding author
Additional information
This paper is part of a themed issue on synthetic and natural photoswitches.
Rights and permissions
About this article
Cite this article
Nack, M., Radu, I., Gossing, M. et al. The DC gate in Channelrhodopsin-2: crucial hydrogen bonding interaction between C128 and D156. Photochem Photobiol Sci 9, 194–198 (2010). https://doi.org/10.1039/b9pp00157c
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1039/b9pp00157c