The role of C621 and C665 in rapid binding and activation of hTRPA1. (A) Normalized 10 µM B-IA (60 s) binding to hTRPA1 WT (white bars; n = 7) and C621A mutant (red bars; n = 4). (B) Normalized 10 µM B-IA (60 s) binding to hTRPA1 WT (white bars; n = 5) and C665L mutant (green bars; n = 6). (A and B) The dollar sign denotes difference from WT ($, P < 0.05). Asterisks denotes difference from control (clear bars) with IA pretreatment (100 µM, 10 min; hatched bars; *, P < 0.05). Bands in the blots are 131 kD. (C) Activation of TRPA1 constructs (WT, C621A, and C665L) by vehicle (Veh), 30 µM IA, and 200 µM thymol (n ≥ 49). (A–C) Error bars denote SEM.