Assessing the Role of R120 in the Gating of CrChR2 by Time-Resolved Spectroscopy from Femtoseconds to Seconds

The light-gated ion channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) is the most frequently used optogenetic tool in neurosciences. However, the precise molecular mechanism of the channel opening and the correlation among retinal isomerization, the photocycle, and the channel activity of the protein are missing. Here, we present electrophysiological and spectroscopic investigations on the R120H variant of CrChR2. R120 is a key residue in an extended network linking the retinal chromophore to several gates of the ion channel. We show that despite the deficient channel activity, the photocycle of the variant is intact. In a comparative study for R120H and the wild type, we resolve the vibrational changes in the spectral range of the retinal and amide I bands across the time range from femtoseconds to seconds. Analysis of the amide I mode reveals a significant impairment of the ultrafast protein response after retinal excitation. We conclude that channel opening in CrChR2 is prepared immediately after retinal excitation. Additionally, chromophore isomerization is essential for both photocycle and channel activities, although both processes can occur independently.