期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 116, 期 19, 页码 9380-9389出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1818707116
关键词
channelrhodopsin-2; optogenetics; time-resolved FTIR; electrophysiology; photoisomerization
资金
- Deutsche Forschungsgemeinschaft (DFG) [SPP 1926]
- DFG [SFB1078]
- Cluster of Excellence Unifying Concepts in Catalysis Berlin International Graduate School of natural Science and Engineering (BIG-NSE)
- Hertie Foundation
Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photo current 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 P-480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P-480 induces a parallel syn-photocycle with an open channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P-480 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.
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