The crystal structure of Gt ACR1 from Guillardia theta revealed an intramolecular tunnel predicted to expand to form the anion-conducting channel upon photoactivation (Li et al. 2019). The location of the retinylidene photoactive site within the tunnel raised the question of whether, in addition to triggering channel opening by photoisomerization, the site also participates in later channel processes. Here we demonstrate the involvement of the photoactive site in chloride conductance and channel closing. Electrostatic perturbation of the photoactive retinylidene Schiff base region by glutamate substitutions alters the rectification of the photocurrent as well as channel closing kinetics. Substitutions on opposite sides of the photoactive site causes opposite changes, with channel closing kinetically correlated with Schiff base deprotonation, and the extent of these effects closely correlate with distance of the introduced glutamyl residue from the photoactive site.
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