Extended Data Fig. 1: Reversing the fluorescence response.

(A) Peak responses following electroporation of HEK293-Kir2.1 cells transfected with PCR-assembled genes encoding all 400 combinations of 20 amino acids each at positions 150 and 151 in ASAP2f L146G S147T 414Q. Note some responses were negative (dimming) and others were positive (brightening). Mutant 150D 151 G and mutant 150 T 151 T showed the largest positive responses. (B) Left, relative fluorescence changes from –70 mV of HEK293A cells transfected with plasmids expressing mutant 150D 151 G or mutant 150 T 151 T at various command voltages during patch-clamp electrophysiology. Mean responses normalized to –70 mV are plotted. Error bars represent standard error of the mean (SEM) of n = 8 cells in each group. Right, activation kinetics in response to 2-ms and 1-s steps from –70 mV to +30 mV of mutant 150D 151 G or mutant 150 T 151 T. Gray band represents SEM of n = 8 cells in each group. Mutant 150D 151 G was designated ASAP4.0.