Direct targeting of single TRPA1 channels by DNFB
To further confirm DNFB directly acting on single TRPA1 channels, we performed the single-channel recordings in an inside-out patch configuration. As a control, application of TRPA1 agonist 300 µM AITC increased the channel opening levels to level 2 from level 1 with the single channel conductance about 148.37 ± 0.38 pS and channel open probability about to 25.58 % from 0.59 % of basal control (n = 9-11, P < 0.05) (Fig. 4a, b and c). Adding DNFB at 300 µM resulted in a significant increase of the channel opening from level 1 to level 4 with single channel conductance about 153.5 ± 0.05 pS and channel open probability to 33.25 % (n = 6-14,P < 0.001) from 0.59% (Fig. 4a, b and c). These results confirmed that DNFB activates TRPA1 currents by directly acting on single channels through increase of channel open probability.