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	Figure 1. Species-specific differences in HC antagonism.(a,b) Representative traces of wt-hTRPA1 (a) or wt-fTRPA1 (b) currents in response to repeated CA application in X. laevis oocytes. (c,d) Representative traces showing that 50 μM HC inhibited CA-evoked currents in wt-hTRPA1 (c), but not in wt-fTRPA1 (d). (e,f) The differences in the inhibitory effects of HC (10, 25 and 50 μM) on CA-induced currents in oocytes expressing wt-hTRPA1 (e) or wt-fTRPA1 (f). Normalized current: ratio of the current amplitude in the second CA stimulation to that of the first with or without HC. (**P < 0.01), one way ANOVA post hoc Tukey test. Data are shown as the mean ± SEM. (e) wt-hTRPA1 (CA, n = 17; CA + 10 μM HC, n = 20; CA + 25 μM HC, n = 18; CA + 50 μM HC, n = 17). (f) wt-fTRPA1 (CA, n = 12; CA + 10 μM HC, n = 7; CA + 25 μM HC, n = 13; CA + 50 μM HC, n = 14).
	Figure 2. Effects of HC on various chimeric fTRPA1 and hTRPA1 channels.(a) Schematic representation of the hTRPA1 and fTRPA1 chimeras. hTRPA1 and fTRPA1 are depicted in grey and white, respectively. The regions of hTRPA1 included in the chimeras are indicated by brackets. (b–f) The effects of HC at different concentrations on the CA-evoked currents. Normalized current: ratio of the current amplitude in the second CA stimulation to that of the first with or without HC. (**P < 0.01), one way ANOVA post hoc Tukey test. Data are shown as the mean ± SEM. (b) F-H (T1-Ct) (0.3 mM CA, n = 11; CA + 10 μM HC, n = 9; CA + 25 μM HC, n = 9; CA + 50 μM HC, n = 10). (c) F-H (T1-T6)-F (0.5 mM CA, n = 16; CA + 10 μM HC, n = 6; CA + 25 μM HC, n = 12; CA + 50 μM HC, n = 11). (d) F-H (T3-Ct) (0.5 mM CA, n = 15; CA + 10 μM HC, n = 5; CA + 25 μM HC, n = 10; CA + 50 μM HC, n = 8). (e) F-H (T5-Ct) (0.2 mM CA, n = 7; CA + 10 μM HC, n = 5; CA + 25 μM HC, n = 7; CA + 50 μM HC, n = 8). (f) F-H (Ct) (0.1 mM CA, n = 12; CA + 10 μM HC, n = 5; CA + 25 μM HC, n = 5; CA + 50 μM HC, n = 13).
	Figure 3. Identification of the amino acid responsible for the inhibitory effects of HC on hTRPA1.(a) Amino acid alignment of the regions delineated by the chimeric analysis shown in Fig. 2. Amino acids were examined using the following formula: (human TRPA1 ≈ mouse TRPA1 ≈ green anole TRPA1 ≈ chicken TRPA1) ≠ (western clawed (WC) frog TRPA1); these differences are highlighted in black. The arrow indicates the amino acid involved in the inhibitory effect of HC. (b–d) Differences in the inhibitory effects of HC (b and c) or A96 (d) at different concentrations on CA-evoked currents in wt-hTRPA1 or hTRPA1-N855S. Normalized current: ratio of the current amplitude in the second CA stimulation to that of the first with or without HC. Each bar represents the mean ± SEM. (**P < 0.01), one way ANOVA post hoc Tukey test or t-test. (b) wt-hTRPA1 and hTRPA1-N855S (0.3 mM CA, n = 5,5; CA + 0.1 μM HC, n = 6,6; CA + 1 μM HC, n = 7,6; CA + 3 μM HC, n = 6,6; CA + 10 μM HC, n = 6,6; CA + 25 μM HC, n = 6,7; CA + 50 μM HC, n = 5,7). (c) wt-hTRPA1 and hTRPA1-N855S (0.3 mM CA, n = 11,11; CA + 0.1 μM A96, n = 4,5; CA + 1 μM A96, n = 4,5).
	Figure 4. Effects of HC on the hTRPA1-N855S mutant expressed in HEK293T cells.(a,b) The effects of HC (20 μM) on the CA (0.1 mM)-evoked increase in [Ca2+]i in HEK293 cells expressing wt-hTRPA1 or hTRPA1-N855S. Each bar represents the mean ± SEM. (*P < 0.05), t-test. (c) Comparison of the normalized ratio to the ionomycin responses between wt-hTRPA1 (n = 5 with and 5 without HC, respectively) and hTRPA1-N855S (n = 7 with and 7 without HC, respectively).
	Figure 5. HC inhibitory effects on fTRPA1 mutants.(a,b) Representative traces of CA (0.5 mM)-evoked currents in X. laevis oocytes expressing fTRPA1-S880N without (a) or with (b) HC (50 μM). (c,d) The effects of HC (c) or A96 (d) at different concentrations on CA-evoked currents in wt-fTRPA1 and fTRPA1-S880N. Normalized current: ratio of the current amplitude in the second CA stimulation to that of the first with or without HC. Each bar represents the mean ± SEM. (**P < 0.01), t-test. (c) (CA, n = 12,11; CA + 25 μM HC, n = 5,6; CA + 50 μM HC, n = 7,9). (d) (CA, n = 12,11; CA + 0.1 μM A96, n = 6,5; CA + 1 μM A96, n = 5,7).
	Figure 6. Effects of TRPA1 antagonists on zTRPA1b.(a) Comparison of N855 in hTRPA1 (asterisk) with that in mouse, chicken, green anole, western clawed (WC) frog, and zebrafish TRPA1. (b,c) Representative traces of CA-evoked currents in wt-zTRPA1b in the presence of 50 μM HC (b) or 1 μM A96 (c). (d,e) Effects of HC (d) or A96 (e) at different concentrations on CA (1 mM)-evoked wt-zTRPA1b currents. Normalized current: ratio of the current amplitude in the second CA stimulation to that of the first with or without HC. Each bar represents the mean ± SEM. (d) (CA, n = 18; CA + 10 μM HC, n = 11; CA + 25 μM HC, n = 11; CA + 50 μM HC, n = 13). (e) (CA, n = 18; CA + 0.1 μM A96, n = 10; CA + 1 μM A96, n = 10).
	Figure 7. Changes in the inhibitory effects of HC by a single point mutation in hTRPA1 and zTRPA1b.(a–d) Effects of HC (a,c) or A96 (b,d) at different concentrations on CA-evoked currents in wt-hTRPA1 and hTRPA1-N855R (a,b) or on the CA-evoked currents in wt-zTRPA1b and zTRPA1b-R860N (c,d). Normalized current: ratio of the current amplitude in the second CA stimulation to that of the first with or without HC. Each bar represents the mean ± SEM. (**P < 0.01, *P < 0.05), t-test. (a) wt-hTRPA1 and hTRPA1-N855R (0.3 mM CA, n = 9,9; CA + 25 μM HC, n = 8,8; CA + 25 μM HC, n = 7,8; CA + 50 μM HC, n = 7,8). (b) wt-hTRPA1 and hTRPA1-N855R (0.3 mM CA, n = 9,9; CA + 0.1 μM A96, n = 4,5; CA + 1 μM A96, n = 4,4). (c) wt-zTRPA1b and zTRPA1b-R860N (1 mM CA, n = 5,6; CA + 10 μM HC, n = 5,6; CA + 25 μM HC, n = 5,6; CA + 50 μM HC, n = 5,6). (d) wt-zTRPA1b and zTRPA1b-R860N (1 mM CA, n = 5,6; CA + 0.1 μM A96, n = 4,4; CA + 1 μM A96, n = 4,4).
	Figure 8. Effects of HC on point mutations in the C-terminal region of hTRPA1.(a) Amino acid alignment of the C-terminal regions of TRPA1 from different species. Amino acids were evaluated using the following formula: (human TRPA1 ≈ mouse TRPA1 ≈ green anole TRPA1 ≈ chicken TRPA1) ≠ (western clawed (WC) frog TRPA1); these differences are highlighted in black. (b) Effects of 50 μM HC on CA-evoked currents. Average normalized currents for CA with 50 μM HC were divided by the average normalized current for CA alone. For each mutant, n = 3–5.
	Figure 9. Synergistic interaction between N855 and the C-terminus of TRPA1 on the inhibitory effects of HC.(a,b) Representative traces of CA (0.1 mM)-evoked currents in X. laevis oocytes expressing the F-H (Ct) chimera with the fTRPA1-S880N mutation [F-H (Ct) + S880N] (a) or the F-H (Ct) chimera alone (b). (c,d) Effects of 50 μM HC on CA-evoked currents in the F-H (Ct) + S880N (a) or F-H (Ct) chimera alone (b). (e,f) Effects of HC (c) or A96 (d) at different concentrations on CA-evoked currents in the F-H (Ct) + S880N or F-H (Ct) chimera alone. Normalized current: ratio of the current amplitude in the second CA stimulation to that of the first with or without HC. Each bar represents the mean ± SEM. (**P < 0.01), t-test. (e) F-H (Ct) + fTRPA1-S880N and F-H (Ct) (0.1 mM CA, n = 12,12; CA + 0.1 μM HC, n = 6,4; CA + 1 μM HC, n = 5,5; CA + 3 μM HC, n = 6,4; CA + 10 μM HC, n = 9,8; CA + 25 μM HC, n = 9,9; CA + 50 μM HC, n = 9,9). (f) F-H (Ct) + fTRPA1-S880N and F-H (Ct) (0.1 mM CA, n = 8,8; CA + 1 μM A96, n = 3,3).
	Figure 10. Molecular dynamics simulation confirming the stable bond between HC and N855.(a) Snapshots of the MD simulation at 0 ns (left) and at 100 ns (right). The HC molecule and amino acid residue N855 are shown in a stick representation. These snapshots were rendered using the RasMol graphic software51. (b) A time series of the distance between the O atom of HC and the Hδ atom of N855 in the MD simulation. (c) Highlighted hydrophobic (green) and hydrophilic (purple) regions of the HC molecule.

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