The Pore-Domain of TRPA1 Mediates the Inhibitory Effect of the Antagonist 6-Methyl-5-(2-(Trifluoromethyl)Phenyl)-1H-Indazole

Abstract

The pore-domain of TRPA1 mediates the inhibitory effect of the antagonist 6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole The ion channel TRPA1 gives to the organisms the ability to detect noxious chemicals to sensory neurons and as a result of this, mediates chemical nociception in vivo. Mouse TRPA1 is activated by electrophilic compounds such as mustard-oil (MO) and several physical stimuli such as cold temperature. Both stimuli are mechanistic linked to the N terminus of the channel. Due to its sensory function, the inhibition of TRPA1 activity might provide an effective treatment against chronic and inflammatory pain, for that reason, TRPA1 has become an important target for the development of new and better 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 MO. We identified five mutations located within the pore domain that cause loss of inhibition by Compound 31, one of them in the residue T877 placed in the TM5, important in the binding of menthol, a dose-dependent agonist/blocker. 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.