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Proc Natl Acad Sci U S A. 2019 May 7;116(19):9380-9389. doi: 10.1073/pnas.1818707116. Epub 2019 Apr 19.

Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2.

Author information

1
Department of Biophysics, Ruhr-Universität Bochum, 44780 Bochum, Germany.
2
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.
3
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, 10115 Berlin, Germany hegemann@rz.hu-berlin.de klaus.gerwert@bph.rub.de.
4
Department of Biophysics, Ruhr-Universität Bochum, 44780 Bochum, Germany; hegemann@rz.hu-berlin.de klaus.gerwert@bph.rub.de.

Abstract

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

KEYWORDS:

channelrhodopsin-2; electrophysiology; optogenetics; photoisomerization; time-resolved FTIR

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