期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2019
卷号：116
期号：19
页码：9380-9389
DOI：10.1073/pnas.1818707116
出版社：The National Academy of Sciences of the United States of America
摘要：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.
