Oral Presentation 18th International Congress on Photobiology 2024

Channelrhodopsins (ChR) are light-gated ion channels that passively transport various cations and anions in a light-dependent manner. While these proteins are widely used in optogenetics to optically control the neural activity in vivo, their channel gating mechanism has not been clarified yet. Recent quantum mechanics/molecular mechanics (QM/MM) calculation and time-resolved X-ray crystallography observed that the structure of the retinal chromophore in C1C2, which is a chimeric ChR constructed by combining the amino acid sequences of CrChR1 and CrChR2 found in Chlamyidomonas reinhardtii, is highly distorted in the pre-open state of the protein [1,2]. This structural change in the retinal chromophore is considered to probably facilitate the subsequent channel opening. Here, we studied the photoreaction dynamics of C1C2 by transient absorption spectroscopy, laser patch-clamp, and time-resolved resonance Raman spectroscopy. As previous studies suggested, an increase in the hydrogen-out-of-plane modes indicating the larger twisting of the retinal chromophore compared to the ground state was observed in the photointermediate states. This twisting reached maximum in the P_2b state, which was identified as the full-open state through the laser patch-clamp measurement. The retinal twisting is relaxed in the subsequent P_3b state, and this process is rate-limited by a H⁺ transfer from the protein moiety to the Schiff-base linkage of the retinal chromophore. Our result indicates that the twisting of the polyene chain of the retinal chromophore, which is not observed in other types of microbial rhodopsins, induces the opening of the channel pore in C1C2 [3]. In this presentation, I will present the results of other ChRs and ion pumping rhodopsins and the diversity of the ion-transporting mechanisms in microbial rhodopsins.