Proton activation does not alter antagonist interaction with the capsaicin-binding pocket of TRPV1.

Vanilloid receptor 1 (TRPV1) is activated by chemical ligands (e.g., capsaicin and protons) and heat. In this study, we show that (2 E )-3-[2-piperidin-1-yl-6-(trifluoromethyl)pyridin-3-yl]- N -quinolin-7-ylacrylamide (AMG6880), 5-chloro-6-{(3 R )-3-methyl-4-[6-(trifluoromethyl)-4-(3,4,5-trifluorophenyl)-1 H -benzimidazol-2-yl]piperazin-1-yl}pyridin-3-yl)methanol (AMG7472), and N -(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2 H )-carboxamide (BCTC) are potent antagonists of rat TRPV1 activation by either capsaicin or protons (pH 5) (defined here as group A antagonists), whereas (2 E )-3-(6- tert -butyl-2-methylpyridin-3-yl)- N -(1 H -indol-6-yl)acrylamide (AMG0610), capsazepine, and (2 E )-3-(4-chlorophenyl)- N -(3-methoxyphenyl)acrylamide (SB-366791) are antagonists of capsaicin, but not proton, activation (defined here as group B antagonists). By using capsaicin-sensitive and insensitive rabbit TRPV1 channels, we show that antagonists require the same critical molecular determinants located in the transmembrane domain 3/4 region to block both capsaicin and proton activation, suggesting the presence of a single binding pocket. To determine whether the differential pharmacology is a result of proton activation-induced conformational changes in the capsaicin-binding pocket that alter group B antagonist affinities, we have developed a functional antagonist competition assay. We hypothesized that if group B antagonists bind at the same or an overlapping binding pocket of TRPV1 as group A antagonists, and proton activation does not alter the binding pocket, then group B antagonists should compete with and prevent group A antagonism of TRPV1 activation by protons. Indeed, we found that each of the group B antagonists competed with and prevented BCTC, AMG6880 or AMG7472 antagonism of rat TRPV1 activation by protons with p A 2 values similar to those for blocking capsaicin, indicating that proton activation does not alter the conformation of the TRPV1 capsaicin-binding pocket. In conclusion, group A antagonists seem to lock the channel conformation in the closed state, blocking both capsaicin and proton activation.