Fig. 3

FK506 activates human TRPA1. a A representative trace of a whole-cell patch-clamp recording in a HEK293T cell expressing human TRPA1 (hTRPA1) exposed to 55 μM FK506 (black bar) and 100 μM AITC (white bar). Membrane potential was held at − 60 mV and ramp pulses from − 100 to +100 mV were applied every 3 s. Insets indicate the representative I–V curves of the hTRPA1 currents in the absence (a) or presence (b) of 55 μM FK506. Allyl isothiocyanate (AITC) was used as a positive control for TRPA1. b A TRPA1 antagonist (3 μM A967079) inhibited a 55 μM FK506-induced current in a HEK293T cell expressing hTRPA1. c, d Summary of the peak currents at − 60 mV (c) or + 100 mV (d) induced by 30 μM or 55 μM FK506 in hTRPA1- or mock-transfected HEK293T cells. Each column represents the mean + SEM from 8–9 cells. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t test with Bonferroni correction. *, P < 0.05; **, P < 0.01 vs. mock. e Representative traces of inside-out single channel recordings in HEK293T cells expressing hTRPA1 in the absence (basal) or presence of 55 μM FK506, or 20 μM AITC. Dotted lines indicate 0 current levels (c). Membrane potential was held at + 30 mV. f NPo values were calculated in the single-channel currents. Ordinates: NPo (channel number × open probability). Each column represents the mean + SEM from four experiments. Statistical significance was assessed using ANOVA followed by a two-tailed multiple t test with Bonferroni correction. *; P < 0.05, **; P < 0.01 vs. basal