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. In this study, we show that (2E)-3-[2-piperidin-1-yl-6-(trifluoromethyl)pyridin-3-yl]-Nquinolin-7-ylacrylamide (AMG6880), 5-chloro-6-{(3R)-3-meth- and N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) are potent antagonists of rat TRPV1 activation by either capsaicin or protons (pH 5) (defined here as group A antagonists), whereas (2E)-3-(6-tertbutyl-2-methylpyridin-3-yl)-N-(1H-indol-6-yl)acrylamide (AMG-0610), capsazepine, and (2E)-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 pA 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.

A Polyclonal Antibody to the Prepore Loop of Transient Receptor Potential Vanilloid Type 1 Blocks Channel Activation

“…We previously postulated that TRPV1 antagonists acting through the capsaicin-binding pocket fall into two distinct categories (Gavva et al, 2005c): class A compounds block channel activation both by capsaicin and protons, whereas class B compounds selectively abolish capsaicin activation. The results presented here indicate that the rabbit anti-rat TRPV1 polyclonal antibody (Ab-156H) binding at a site outside of the capsaicin-binding pocket acts as a full antagonist of proton activation and a partial antagonist of capsaicin, anandamide and heat activation, thus representing a class A antagonist with an unusual profile.…”

“…Molecular determinants for proton activation have been reported to be present in the prepore loop, such as Glu 600 and Glu 648 (Jordt et al, 2000) and are different from those that constitute the vanilloidbinding pocket. Competitive antagonists of TRPV1, such as AMG6880, AMG7472, and BCTC that interact at the vanilloid-binding pocket, seem to lock the channel conformation in the closed state to block all modes of activation (Gavva et al, 2005c). Ruthenium red, a nonselective pore blocker, acts as an antagonist of all modes of TRPV1 activation via interaction with residues in the channel pore, such as Asp 646 (Garcia-Martinez et al, 2000).…”

Human Sensory Neuron-specific Mas-related G Protein-coupled Receptors-X1 Sensitize and Directly Activate Transient Receptor Potential Cation Channel V1 via Distinct Signaling Pathways

“…However, Tyr-511 is not a part of the TRPV1 binding site for protons (47), and proton binding to the TRPV1 does not interfere with its binding to CAP (48). To investigate a role of Tyr-511 for TRPV1 activation by CAP, OAG, protons, BAM8 -22, and BK, we replaced tyrosine 511 by an alanine (TRPV1-Y511A-YFP).…”

TRPV1 Antagonists as Analgesic Agents

“…However, not unexpectedly, some of these new TRPV1 blockers turned out to be stimulus-specific whereas others appear to block several means of activation [92,93]. For instance, AMG0610 (from Amgen) and SB-366791 (developed by GlaxoSmithKline) inhibit the activation of rat TRPV1 by capsaicin but not by acid, whereas I-RTX, BCTC, AMG6880, AMG7472, AMG9810 and A-425619 are TRPV1 antagonists that do not differentiate between capsaicin and protons [92,[94][95][96].…”

“…For instance, AMG0610 (from Amgen) and SB-366791 (developed by GlaxoSmithKline) inhibit the activation of rat TRPV1 by capsaicin but not by acid, whereas I-RTX, BCTC, AMG6880, AMG7472, AMG9810 and A-425619 are TRPV1 antagonists that do not differentiate between capsaicin and protons [92,[94][95][96]. Compound AMG8562 does not block heat-evoked activation of rat TRPV1 [93].…”