K620 is required for high reactivity of C621. (A) Normalized 10 µM B-IA (60 s) binding to hTRPA1 at pH 7.4 (green bars) and 5.0 (red bars) without (clear bars) and with (hatched bars) IA pretreatment (100 µM, 10 min at pH 7.4; n ≥ 3). Dollar signs denote difference from pH 7.4 with pH 5.0 ($, P < 0.05). Asterisks denote difference from control with IA pretreatment (*, P < 0.05). IA pretreatment data were subtracted from control to show reactive binding (crosshatched bars). (B) Predicted structure of C621 domain based on Paulsen et al. (2015). (C) Modeling of the change in Cys deprotonation energies (ΔΔE) caused by proximity of CH₄, NH₄⁺, and HCOO⁻ groups. (D) Normalized 10 µM B-IA (60 s) binding to hTRPA1 WT (white bars, n = 6) and K620A mutant (blue bars, n = 8). The dollar sign denotes difference from WT ($, P < 0.05). Asterisks denote difference from control (clear bars) with IA pretreatment (100 µM, 10 min; hatched bars; *, P < 0.05). (A and D) All bands in the blots are 131 kD. (E) Activation of TRPA1 constructs (WT, C621A, and K620A) by vehicle (Veh), 30 µM IA, and 200 µM thymol (n ≥ 124). (F) Activation of TRPA1 constructs (WT, C621A, and K620A) by vehicle (Veh), 100 µM H₂O₂, 100 µM AITC, and 200 µM thymol (n ≥ 33). (A and D–F) Error bars denote SEM.