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

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-<i>trans</i>,C=N-<i>anti</i> conformation only. Upon light activation, a branching reaction into either a 13-<i>cis</i>,C=N-<i>anti</i> or a 13-<i>cis</i>,C=N-<i>syn</i> retinal conformation occurs. The <i>anti</i>-cycle features sequential H⁺ and Na⁺ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-<i>cis</i>,C=N-<i>syn</i> 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 <i>syn</i>-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-<i>syn</i> cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel <i>anti</i>- and <i>syn</i>-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.