The single-point mutation R721A inverts the selectivity of TRPV1 for PI(4,5)P₂ and PI(4)P. (A) Current traces for activation of TRPV1-R721A channels by PI(4,5)P₂ at the indicated concentrations. (B) Current traces for activation of TRPV1-R721A by PI(4)P. (A and B) 1 µM capsaicin was present for all measurements except for the current shown as the dotted trace, which is the leak current in the absence of capsaicin. This leak current was not subtracted from the other traces. The voltage protocol involved holding patches at 0 mV, followed by voltage jumps to −100 mV and 100 mV, followed by a return to 0 mV. PolyK was applied to the patch before acquisition of traces shown. (C) Capsaicin dose–response curves for TRPV1-WT (solid line and closed circles) and TRPV1-R721A (dashed line and open circles). For TRPV1-WT, the EC₅₀ value was 79.5 nM (n_H = 1, n = 4–5 patches). For TRPV1-R721A, the EC₅₀ value for capsaicin was 330 nM (n_H = 1, n = 4). (D) Normalized current in TRPV1-WT (solid, closed) and TRPV1-R721A (dashed, open) versus solution concentration of PI(4,5)P₂ or PI(4)P. For TRPV1-WT, the EC₅₀ values were 0.68 µM (n = 4–7 patches) and 20.9 µM (n = 5 patches) for PI(4,5)P₂ and PI(4)P, respectively, and n_H values obtained from the Hill fits were 2 for both curves. For TRPV1-R721A, EC₅₀ values were 0.88 µM (n_H = 2, n = 3–5) and 0.144 µM (n_H = 2, n = 4) for PI(4,5)P₂ and PI(4)P, respectively. (E) Same as D, but the data and fitted model are plotted versus mole fraction of lipids in the plasma membrane cytosolic leaflet. (C–E) SEM is shown.