Structural Changes in the Retinal Chromophore and Ion-Conducting Pathway of an Anion Channelrhodopsin GtACR1

Abstract

Guillardia theta anion-conducting channelrhodopsin 1 (GtACR1) is a light-gated anion channel and is widely used as a representative optogenetic tool for neuronal silencing. However, its gating mechanism is unclear due to the lack of insight into its structural changes and kinetics. Here, we measured the structural changes in the retinal chromophore of GtACR1 throughout its photocycle using time-resolved resonance Raman spectroscopy. Moreover, we investigated the rate-limiting factors of the gating through transient absorption spectroscopy and laser patch clamp electrophysiology. Our results provided the following understanding: the retinal chromophore keeps planar structure even in the open state. The ion-conducting pathway extends to the retinal Schiff base (RSB) as the channel opens. The channel closes in a two-step process, rate-limited by the RSB deprotonation and reprotonation. These findings reveal that the gating mechanism of GtACR1 differs markedly from that of a cation channelrhodopsin C1C2 and contribute to the development of next-generation optogenetic tools.