Cells were perfused with AITC (100 μM) or cinnamaldehyde (500 μM) solution for 20 seconds in all experiments. (A) Electrophysiological response of a representative transfected HEK cell to AITC in the absence or presence of ruthenium red (RR; 100 μM). Voltage ramps from –120 to +100 mV (340 ms) were applied. The inset graph shows AITC-activated current density in the absence or presence of ruthenium red (**P < 0.001). (B) AITC-activated inward currents underwent strong tachyphylaxis, giving much smaller responses on repeated applications of AITC. The inset graph shows normalized currents in each AITC challenge (n = 6). Currents were normalized to the currents evoked initially by AITC. (C) AITC-activated currents were sensitized after perfusion for 60 seconds with solution containing 100 μM SL-NH2. However, this sensitization was not sustainable 180 seconds after SL-NH2 application. (D) SL-NH2–mediated potentiation of AITC-activated currents. AITC was reapplied 60 seconds after exposure to bath solution with or without SL-NH2 (SL; 10, 50, or 100 μM) or LR-NH2 (LR). Currents were normalized to values first induced by AITC application in the absence of SL-NH2 or LR-NH2. Cont, control group (preperfused with bath solution without SL-NH2 before reapplication of AITC). (E) Cinnamaldehyde-evoked TRPA1 currents were potentiated by SL-NH2 but not LR-NH2. (F) AITC-activated currents were potentiated by application of trypsin (50 nM). Holding potential (V_h) was –60 mV in all experiments. Numbers in parentheses indicate cells tested. *P < 0.05 versus control group; ^#P < 0.05 versus LR-NH2; unpaired Student’s t test.