Figure 6.

LE135 directly activates TRPA1 channels. LE135 (1 and 10 μM) activated an outward current at +60 mV and an inward current at −60 mV in a TRPA1-expressing HEK293T cell. Current traces in (A) show representative current–voltage relationships of LE135-activated currents. The time course of the current is shown in (B). (C) Concentration–response curve of LE135-activated inward currents taken at −60 mV in TRPA1-expressing HEK293T cells is fitted with the logistic equation as described in Figure 1. The graph inset illustrates the maximal responses (current densities) evoked by saturating concentrations of LE135 (100 μM) and AITC (100 μM). Representative pictures (D) and traces (E) illustrate that LE135-elicited intracellular Ca²⁺ responses are abolished in Trpv1^−/− DRG neurons pre-treated with TRPA1 antagonist HC030031 (upper panel) or Trpa1^−/− DRG neurons pre-treated with TRPV1 antagonist AMG9810 (lower panel). Each trace corresponds to the change of fluorescence ratio in a single neuron. (F) Percentage of DRG neurons responding to LE135, capsaicin (CAP), AITC and KCl in neurons isolated from Trpv1^−/− or Trpa1^−/− mice (n ≥ 350 per genotype).