The Pore-Domain of TRPA1 Mediates the Inhibitory Effect of the Antagonist 6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole

The transient receptor potential ion channel TRPA1 confers the ability to detect tissue damaging chemicals to sensory neurons and as a result mediates chemical nociception in vivo. Mouse TRPA1 is activated by electrophilic compounds such as mustard-oil and several physical stimuli such as cold temperature. Due to its sensory function inhibition of TRPA1 activity might provide an effective treatment against chronic and inflammatory pain. Therefore, TRPA1 has become a target for the development of analgesic drugs. 6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (Compound 31) has been identified by a chemical screen and lead optimization as an inhibitor of chemical activation of TRPA1. However, the structures or domains of TRPA1 that mediate the inhibitory effect of Compound 31 are unknown. Here, we screened 12,000 random mutant clones of mouse TRPA1 for their sensitivity to mustard-oil and the ability of Compound 31 to inhibit chemical activation by mustard-oil. In addition, we separately screened this mutant library while stimulating it with cold temperatures. We found that the single-point mutation I624N in the N-terminus of TRPA1 specifically affects the sensitivity to mustard-oil, but not to cold temperatures. This is evidence that sensitivity of TRPA1 to chemicals and cold temperatures is conveyed by separable mechanisms. We also identified five mutations located within the pore domain that cause loss of inhibition by Compound 31. This result demonstrates that the pore-domain is a regulator of chemical activation and suggests that Compound 31 might be acting directly on the pore-domain.