Val109 and Ile170 contribute to channel function in Guillardia theta-derived anion channelrhodopsins

Anion channelrhodopsins (ACRs) are widely used for optical silencing of neurons. Photocurrents and kinetics are crucial for the precise control of cell hyperpolarization. In this study, we investigated the roles of amino acid residues in each transmembrane domain of the Guillardia theta-derived anion channelrhodopsins, GtACR1 and GtACR2, to develop high-performance ACRs. Several chimaera ACRs were generated by replacing the transmembrane (TM) domains of GtACR1 with homologous counterparts of GtACR2, and photocurrents in HEK cells transduced with each variant were recorded by patch-clamp. The relationship between photocurrent and protein localisation was determined. Chimaeras of TM4, TM5, and TM6 in GtACR1 replaced with the homologous counterparts of GtACR2 showed wavelength changes and fast kinetics(τ_on/off). V109 of TM3, the putative ion tunnel with I170, one of the key interactions for the peak photocurrent. Molecular dynamics simulations using data from the point mutation analysis in I170 showed that the distance from V109 to I170 correlated negatively with the peak photocurrents. The relationship between τ-off and the distance from E68 to N239, which formed the bottleneck of the ion tunnel, was positively correlated. These findings provide useful information for the development of ACRs with higher light sensitivities and faster kinetics(τ_on/off).