Characterization of the tail current of Channelrhodopsin-2 variants

Our study focused on specific ChR2 variants, particularly those with the Step function Opsins (SFO) mutation at the D156-C128 gate. These are widely used in optogenetics due to their heightened sensitivity to light and bi-stable prolonged activation. However, in some ChR2 variants, specifically D156 mutants, a tail current occurs when continuous light exposure is stopped. We specifically examined the D156H-T159S ChR2 variant, which demonstrated a tail current that was somewhat responsive to light and voltage, with a single-channel current of around 9fA, similar to wt-ChR2 as determined by stationary noise analysis. To further investigate, we used nonstationary noise analysis in cell-attached patching mode, which revealed that the tail current's single-channel current falls within the same range as the peak current, albeit with mild contamination from adaptation and desensitization. This finding strongly supports the notion that a portion of the ChR2 molecules open or re-open at the end of illumination, leading to further membrane depolarization.