Extended Data Fig. 5. Characterization of TRPA1 activation by IA and BIA.
a, IA (100 μM) activates TRPA1 through covalent modification of cysteines; AITC (250 or 1000 μM). Data represent n = 6 (WT) or 5 (3C) independent experiments. Statistical significance is reported as the result of an unpaired two-tailed Mann-Whitney test; **, p = 0.002; Vhold = −80 mV. b,c, No single cysteine is sufficient for TRPA1 activation by IA. WT, data represent n = 9 independent experiments ; C621S/C641S n = 3; C621S/C665S, n = 3; and C641S/C665S, n = 4. Data were acquired in whole-cell patch clamp mode and reflect the results of 500 msec. test pulse (80 mV). Vhold = −80 mV. Doses: IA, 100 μM; A-96,10 μM; AITC, 250 or 1000 μM. Scale bars: x, 50 msec.; y, 100 pA. I = 0, dashed line. d, Quantification of double cysteine mutant data. (left) WT, n = 6 independent experiments; C621S/C641S n = 3; C621S/C665S, n = 3; and C641S/C665S, n = 3. Vhold = −80 mV. (right) WT, data represent n = 9 independent experiments; C621S/C641S n = 3; C621S/C665S, n = 3; and C641S/C665S, n = 4. Doses: IA, 100 μM; A-96,10 μM; AITC, 250 or 1000 μM. Statistical significance is reported as the result of a Kruskal-Wallis test with post-hoc Dunn’s test to correct for multiple comparisons; *, p = 0.02; **, p = 0.007. e,f C621S display complete loss of IA sensitivity while C641S retains full sensitivity. Data represent n = 5 independent experiments/construct. Data were acquired in whole-cell patch clamp mode and reflect the results of 500 msec. test pulses from −80 to 80 mV. Vhold = −80 mV. Doses: IA, 100 μM; A-96, 10 μM. Scale bars: x, 25 msec.; y, 100 pA. g, Binding of BIA to TRPA1 C641S/C665S double mutant (C621*) is similar to wildtype. Statistical significance is represented as the results of one-way ANOVA with post-hoc Holm-Sidak correction for multiple comparisons; *, p = 0.03; n = 3 independent experiments/construct. h, TRPA1 cysteine pKa values and deduced proportion of thiolate in the agonist-free state (PDB-ID: 6V9W), and IA-bound (‘Activated,’ PDB-ID: 6V9V) state in the presence or absence of covalent modification at C621. All summary data are displayed as the mean ± S.E.M..