Fig. 5.

hTRPA1 Residues G238, N249, and K270 in AnkyrinR #6 mediate thiadiazole Interaction. (A) Representative steady-state traces of Compound 1 effects on hTRPA1 G238K/N249S/K270N (hTriple) and rTRPA1 K239G/S250N/N271K (rTriple) channels. Channels were activated with 300 µM CA and Compound 1 was applied in the continued presence of agonist. The gray line denotes zero current level, and the voltage protocol is shown in Fig. 2A. (B) Concentration response curves of Compound 1 on hTriple and rTriple channels with WT hTRPA1 and rTRPA1 shown for reference. Potencies with 95% confidence intervals (n = 4–8 per construct): rTriple IC₅₀ = 0.15 μM (95% CI = 0.11–0.19 μM), hTRPA1 = 0.13 µM (95% CI = 0.10–0.16 µM), hTriple IC₅₀ > 10 µM, rTRPA1 IC₅₀ > 10 µM. (C) A-967069 which interacts with the S5 helix of the channel pore is unaffected by the mutations that affect Compound 1 potency. Only the WT TRPA1 fits are shown for clarity (n = 5 per construct): hTRPA1 IC₅₀ = 0.04 µM (95% CI = 0.03–0.05 µM) and rTRPA1 IC₅₀ = 0.20 µM (95% CI = 0.17 = 0.24 µM).