Figure 3. Selectivity activation of TRPA1 currents by DNFB in whole-cell recordings. (a) Whole-cell TRPA1 currents was activated by 5 µM DNFB (green bar) or 300 µM AITC (red bar) before inhibition by TRP channel blocker 20 µM RR (pink bar) (n = 5). (b) TRPV1 current in response to 5 µM DNFB (green bar) or 1 µM TRPV1 agonist capsaicin (red bar) before inhibition by 20 µM RR (pink bar). DNFB displaying no activation on TRPV1 (n = 5). (c) TRPV3 current evoked by 50 µM TRPV3 agonist 2-APB (red bar) or 5 µM DNFB (green bar), DNFB showing no activation (n = 5). (d) TRPV4 current was activated by 5 µM DNFB (green bar) or 1 µM TRPV4 agonist 0.1 µM GSK101 (red bar) before inhibition by 20 µM RR (pink bar), DNFB showing no activation (n= 5). (e) Concentration-dependent activation of TRPA1 currents of different concentrations of DNFB (0.1 µM to 100 µM) (n = 8) (left panel) and AITC (n = 6) (3 µM to 3000 µM) (right panel). (f) Fitting curves by Hill equation for comparing concentration-dependent activation of hTRPA1 currents by DNFB with EC50 of 2.36 ± 0.26 µM (n = 5-8, green) and AITC with EC50 of 17.77 ± 5.02 µM (n = 5-8, red).
Figure 4. Increase of single-channel open probability of TRPA1 by DNFB. (a) Activity in an inside-out patches excised from TRPA1-HEK293 cells held at +80 mV. 300 µM AITC (red bar) and DNFB (green bar) were added as indicated. Shown are raw current trace (upper), in an expanded time scale (middle) and NPo in 500-ms bins (lower). (b) Conductance and amplitude histograms of openings recorded at +80 mV were fitted with Gaussians functions (basal-black line, AITC-red line, DNFB-green line). (c) Summary for calculated mean of POPEN values in the presence of 300 µM DNFB and AITC (n = 6-14). Data are shown as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001, by one-way analysis of variance.