Ion-conducting and gating molecular mechanisms of channelrhodopsin revealed by true-atomic-resolution structures of open and closed states

Abstract

Channelrhodopsins (ChRs) have emerged as major optogenetics tools, particularly in neuroscience. Despite their importance, the molecular mechanism of ChR opening remains elusive. Moreover, all reported structures of ChRs correspond to either a closed or an early intermediate state and lack the necessary level of detail owing to the limited resolution. Here we present the structures of the closed and open states of a cation-conducting ChR, OLPVR1, from Organic Lake phycodnavirus, belonging to the family of viral ChRs solved at 1.1- and 1.3-Å resolution at physiologically relevant pH conditions (pH 8.0). OLPVR1 was expressed in Escherichia coli and crystallized using an in meso approach, and the structures were solved by X-ray crystallography. We also present the structure of the OLPVR1 protonated state at acidic pH (pH 2.5) at 1.4-Å resolution. Together, these three structures elucidate the molecular mechanisms of the channel's opening and permeability in detail. Extensive functional studies support the proposed mechanisms. Channel opening is controlled by isomerization of the retinal cofactor, triggering protonation of proton acceptors and deprotonation of proton donors located in the three gates of the channel. The E51 residue in the core of the central gate (similar to E90 of ChR2 from Chlamydomonas reinhardtii) plays a key role in the opening of the channel. E51 flips out of the gate and towards the proton acceptor D200 (D253 in ChR2 in C. reinhardtii), establishing a hydrogen bond between them. Despite differences in subfamilies of ChRs, they share a common gate-cavity architecture, suggesting that they could have similar general gating mechanisms. These results enabled us to design viral rhodopsin with improved properties for optogenetic applications. The structural data and mechanisms might also be helpful for better understanding other ChRs and their engineering.