Activation of VSP (Dr-VSP) reduces PH probe FRET. (A) Cells were transfected with PIP₂-binding PH probes (PH-PLCδ1) fused to CFP or YFP, Dr-VSP, and KCNQ2 and KCNQ3 channel subunits and recorded in whole cell voltage clamp. (B) Principle of PIP₂ measurement by PH probe FRET (see Results and Fig. S3). (C) Photometry setup. Excitation light was scanned from 300 to 500 nm in 200 ms, every 500 ms, and reflected by a dichroic mirror around 440 and 500 nm. Emission light was separated into channels for CFP emission (480/40 nm) and YFP emission (535/30 nm). Time courses (D, E, F, and H) were acquired simultaneously. (D) CFP emission with 440-nm excitation (CFP_C). (E) YFP emission with 440-nm excitation, corrected for CFP emission at 535/30 nm and for direct excitation of YFP by 440-nm excitation light (YFP_C). (F) YFP emission with 500-nm excitation (YFP_Y). (G) FRETr = YFP_C/CFP_C. (H) Tail current amplitude. Membrane was held at −60 mV and depolarized to −20 mV for 300 ms every 500 ms, except for shaded area where membrane was held at +100 mV for 2 s. Tail currents were measured during slow channel deactivation at −60 mV. (I) Time constants of single-exponential fits to FRETr while membrane was held at +100 mV (onset of VSP effect). A summary of 14 cells is shown. (J) Time constant of single-exponential fits to recovery of FRETr after 2 s at +100 mV. A summary of 12 cells is shown.