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-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-,C=N- conformation only. Upon light activation, a branching reaction into either a 13-,C=N- or a 13-,C=N- retinal conformation occurs. The -cycle features sequential H and Na conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-,C=N- isomer represents a second closed-channel state identical to the long-lived P state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P induces a parallel -photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P and stays deprotonated in the C=N- cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel - and -photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.