Incompatible correlation between receptor-operated TRPC6/7 currents and DAG production. (A) Principle of DAG detection by PKC probe FRET. Increments in FRET caused by the translocation of PKCε-CFP_mse to the plasma membrane were detected by a coexpressed membrane-resident acceptor protein (Mry-YFP_mse) in HEK293 cells. (B) Whole-cell TRPC6 currents (top) and FRET changes caused by DAG increments, “FR^dag” (bottom, green circles), recorded from endogenous muscarinic receptor stimulation with 100 µM CCh. The rise of FRET was fitted to the exponential equation: $FRdag=FRdagmax−ΔFRdag×exp(−t/τFRdag)$ (green solid curve). (C) Traces of currents and FR^dag in M₁R-overexpressing cells with 100 µM CCh. (Left) TRPC6 currents. (Right) TRPC7 currents. Prolonged DAG production was observed. Purple zones indicate inconsistencies between current inactivation and DAG production. The inset in the TRPC7 panel shows FR^dag changes over a longer time scale (300 s). (D) Summary of FR^dag levels at the respective current points observed in the robust receptor stimulation (+M₁R and 100 µM). The black and gray bars denote expression of TRPC6 and TRPC7 channels, respectively. (E) Time courses of initial phase of current increase (Δ10–30%) were plotted against kinetics of DAG production (τFR^dag).