Photoexcitation of the P₄⁴⁸⁰ State Induces a Secondary Photocycle That Potentially Desensitizes Channelrhodopsin‑2

Channelrhodopsins (ChRs) are light-gated cation channels. In spite of their wide use to activate neurons with light, the photocurrents of ChRs rapidly decay in intensity under both continuous illumination and fast trains of light pulses, broadly referred to as desensitization. This undesirable phenomenon has been explained by two interconnected photocycles, each of them containing a nonconductive dark state (D1 and D2) and a conductive state (O1 and O2). While the D1 and O1 states correspond to the dark-state and P₃⁵²⁰ intermediate of the primary all-<i>trans</i> photocycle of ChR2, the molecular identity of D2 and O2 remains unclear. We show that P₄⁴⁸⁰, the last intermediate of the all-<i>trans</i> photocycle, is photoactive. Its photocycle, characterized by time-resolved UV/vis spectroscopy, contains a red-shifted intermediate, I₃⁵³⁰. Our results indicate that the D2 and O2 states correspond to the P₄⁴⁸⁰ and I₃⁵³⁰ intermediates, connecting desensitization of ChR2 with the photochemical properties of the P₄⁴⁸⁰ intermediate

Pre-Gating Conformational Changes in the ChETA Variant of Channelrhodopsin‑2 Monitored by Nanosecond IR Spectroscopy

Light-gated ion permeation by channelrhodopsin-2 (ChR2) relies on the photoisomerization of the retinal chromophore and the subsequent photocycle, leading to the formation (on-gating) and decay (off-gating) of the conductive state. Here, we have analyzed the photocycle of a fast-cycling ChR2 variant (E123T mutation, also known as ChETA), by time-resolved UV/vis, step-scan FT-IR, and tunable quantum cascade laser IR spectroscopies with nanosecond resolution. Pre-gating conformational changes rise with a half-life of 200 ns, silent to UV/vis but detected by IR spectroscopy. They involve changes in the peptide backbone and in the H-bond of the side chain of the critical residue D156. Thus, the P₁⁵⁰⁰ intermediate must be separated into early and late states. Light-adapted ChR2 contains a mixture of all-<i>trans</i> and 13-<i>cis</i> retinal in a 70:30 ratio which are both photoactive. Analysis of ethylenic and fingerprint vibrations of retinal provides evidence that the 13-<i>cis</i> photocycle recovers in 1 ms. This recovery is faster than channel off-gating and most of the proton transfer reactions, implying that the 13-<i>cis</i> photocycle is of minor functional relevance for ChR2