Simultaneous measurement of receptor-operated TRPC6 currents and PI(4,5)P₂ detected by 3-cube FRET. (A) Images of PI(4,5)P₂ sensor expressed in HEK293 cells using a 3-cube filter. Phase-contrast image (top left), YFP channel (F_542(A); top right), CFP channel (F_464(D); bottom left), and FRET channel (F_542(D); bottom right) are shown. (B) Diagram of molecules transfected into HEK293 cells. TRPC6, PI(4,5)P₂ sensor (CFP_mse-PHd [blue box] and YFP_mse-PHd [yellow box]), and M₁R were expressed. The excitation wavelengths, 427 and 504 nm, were alternately illuminated. (C) Typical example of CCh-induced TRPC6 currents (top) and the corresponding FRET changes (middle). The FR (middle; circles) calculated by 3-cube methods can yield near absolute FRET efficiency (E_EFF; right axis). Measured parameters were the duration of Δ10–90% and Δ90–50% of the peak current, the kinetics of FRET decay (τFR), and minimum FR (FR_min). The decline of FRET (FR) was fitted with a single-exponential decay (red solid curve): $FR=FRmin+FRΔ×exp(−t/τFR).$ The bottom panel shows the changes in the fluorescence intensities (a.u.) that passed through the respective filter setting.