A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

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 turn-over electrophysiology, time-resolved step-scan FTIR and Raman spectroscopy of fully dark adapted ChR2. This yields a unifying parallel photocycle model explaining all data: in dark-adapted ChR2, the protonated Schiff base retinal 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 and we show that deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light-adaptation. Parallel anti- and syn-photocycles explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.