Improving positively tuned voltage indicators for brightness and kinetics

The recent positively tuned ASAP4-family voltage indicators feature superior photostability compared to the negatively tuned ASAP3, but signal-to-noise ratios (SNRs) for spike detection were not significantly different. To improve spike detection by positively tuned ASAP indicators, we performed multiple rounds of structure-guided saturation mutagenesis of an ASAP4 template while screening directly for faster responses. Our resulting variants, ASAP6.1 and ASAP6b, demonstrated sufficient dynamic range and kinetics, and reported action potentials in vivo by one-photon and two-photon microscopy with high SNR and temporal resolution. Previously, we developed positively tuned ASAP4-subfamily GEVIs with high photostability1. While these had higher {Delta}F/F0 from -70 mV to +30 mV than the negatively tuned ASAP32 or ASAP5, their F-V curves were spread out over a larger voltage range. In addition, ASAP4b and ASAP4e onset kinetics are biexponential with only a minority component of the response exhibiting a time constant of < 10 ms. Both limitations could be due to steric or electrostatic interactions that cause resistance to upward S4 movement in ASAP4 variants. Thus, we set out to engineer positively tuned GEVIs with faster activation kinetics over ASAP4b and ASAP4e, while maintaining or improving steady-state responsivity.