Effect of a translocatable PI 5-phosphatase on TRPM3 activity in HEK-M3 cells. (A) Fluorescence images showing the translocation of the PI(4,5)P₂ sensor PLCδ₁ PH-GFP (green) in HEK-M3 cells. These cells further expressed an FRB coupled plasma membrane linker (PM-FRB-CFP; not depicted) together with either mRFP-FKBP-5-ptase-dom (top; red) or mRFP-FKBP-only (bottom; red), before and after application of 1 µM rapamycin or 10 µM ionomycin. (B) Mean time course (n = 5 for each condition) of the normalized ratio between the GFP fluorescence in the cell center and the entire cell, as a measure of PLCδ₁ PH-GFP translocation from plasma membrane to cytosol. (C) Two representative intracellular Ca²⁺ traces upon repetitive stimulation with PregS (10 µM) in HEK-M3 cells overexpressing PM-FRB-mRFP and mRFP-FKBP-5-ptase-dom, before and after rapamycin-induced PI(4,5)P₂ breakdown. (D) Same as C, but in HEK cells expressing human TRPM8 instead of TRPM3, repetitively stimulated with menthol (50 µM). (E) Amplitude of the second-fourth calcium transients, normalized to the first calcium transient, for the indicated conditions. P values were obtained by unpaired Student’s t test, comparing with the mRFP-FKBP-only condition. (F) Representative time course of PregS-induced (40 µM) current inhibition upon rapamycin-induced PI(4,5)P₂ breakdown in HEK-M3 cells. (G) Same as F, but in HEK cells expressing human TRPM8 instead of TRPM3, stimulated with menthol (50 µM). (H) Analysis of the effect of rapamycin-induced PI(4,5)P₂ breakdown in HEK cells expressing either TRPM3 or TRPM8. PregS- and menthol-induced currents measured in the presence of rapamycin were normalized to the current measured just before the application of rapamycin. P values were obtained by one-way ANOVA and Bonferroni post-hoc test, comparing with the mRFP-FKBP-only condition or as indicated.