Received for publication June 15, 2005.
Revised August 30, 2005.
Accepted for publication August 30, 2005.

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

Abstract

Vanilloid receptor 1 (TRPV1) is activated by chemical ligands (e.g., capsaicin and protons) and heat. Here we show that AMG6880, AMG7472, and BCTC are potent antagonists of rat TRPV1 activation by either capsaicin or protons (pH 5) (defined here as group A antagonists), whereas AMG0610, capsazepine, and SB-366791 are antagonists of capsaicin, but not proton, activation (defined here as group B antagonists). By utilizing capsaicin-sensitive and insensitive rabbit TRPV1 channels, we show that antagonists require the same critical molecular determinants located in the TM3/4 region to block both capsaicin and proton activation, suggesting the presence of a single binding pocket. To determine if 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 pA₂ 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 appear to lock the channel conformation in the closed state, blocking both capsaicin and proton activation.

Key words: Capsaicin/vanilloid, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches, Receptor binding studies

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