Light-dark Adaptation of Channelrhodopsin C128T Mutant

• Published in: The Journal of biological chemistry 2013-04, Vol.288 (15), p.10451-10458
• Main Authors: Ritter, Eglof, Piwowarski, Patrick, Hegemann, Peter, Bartl, Franz J.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Binding Sites; Channelrhodopsin; Chlorocebus aethiops; Conducting State Formation; COS Cells; Dark Adaptation - physiology; Fourier Transform IR (FTIR); Ion Channels; Molecular Biophysics; Mutation, Missense; Photobiology; Photocycle; Photoreceptors; Retinal Isomerization; Retinaldehyde - genetics; Retinaldehyde - metabolism; Rhodopsin - genetics; Rhodopsin - metabolism; UV Spectroscopy
• ISSN: 0021-9258; 1083-351X
• DOI: 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. Background: Channelrhodopsins are light-gated ion channels of microalgae. Results: By FTIR spectroscopy, we identified three different dark and two photoswitchable light-adapted states of the ChR-C128T mutant. Conclusion: We propose a photocycle model that explains both spectroscopic and electrophysiological data. Significance: Color-dependent equilibria determine the stationary photocurrents in ChR applications (optogenetics).