A red-light gate for a cation channel: The conducting state of channelrhodopsin-1 from Chlamydomonas augustae.

Channelrhodopsins (ChRs) are light-controlled ion channels that have become indispensable tools in the field of optogenetics. Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a phylogenetically early member of this class of proteins with red-shifted absorption and pronounced photocurrent kinetics, but the exact correlation between its photocycle intermediate states and channel conductivity remains to be elucidated. Here, we use time-resolved optical absorption spectroscopy (TROD) in the nanosecond-to-second range for wild-type (WT) CaChR1 and its E169Q and D299N counterion variants. Singular value decomposition and global exponential fitting revealed kinetic complexity, suggesting parallel photocycle pathways and isospectral intermediates. The spectral deconvolution method resolved five fundamental spectral forms (K, L, M, N, and R) present in the kinetics. Analysis of their temporal evolution, combined with published electrophysiological data, allowed us to identify the conductive state. Contrary to the dominant model that associates conductivity with the deprotonated M state, we show that a late, red-shifted intermediate state, spectrally similar to the K state and called the O state, is the conductive state. The time evolution of this O state parallels that of the channel current in the WT and is consistent with the reduced conduction in the E169Q and D299N variants. Our findings establish a unified mechanism for channel gating in microbial rhodopsins, where a red-shifted intermediate state controls conduction, and provide a new framework for the rational design of optogenetic tools.