Identification of Species-specific Determinants of the Action of the Antagonist Capsazepine and the Agonist PPAHV on TRPV1

Abstract The vanilloid receptor 1 (VR1 or TRPV1) ion channel is activated by noxious heat, low pH and by a variety of vanilloid-related compounds. The antagonist, capsazepine is more effective at inhibiting the human TRPV1 response to pH 5.5 than the rat TRPV1 response to this stimulus. Mutation of rat TRPV1 at three positions in the S3 to S4 region, to the corresponding human amino acid residues I514M, V518L, and M547L decreased the IC50 values for capsazepine inhibition of the pH 5.5 response from >10,000 nm to 924 ± 241 nm in [Ca2+]i assays and increased capsazepine inhibition of the capsaicin response to levels seen for human TRPV1. We have previously noted that phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) is a strong agonist of rat TRPV1 but not human TRPV1 in [Ca2+]i assays (1). Mutation of methionine 547 in S4 of rat TRPV1 to leucine, found in human TRPV1 (M547L), reduced the ability of PPAHV to activate TRPV1 by ∼20-fold. The reciprocal mutation of human TRPV1 (L547M) enabled the human receptor to respond to PPAHV. These mutations did not significantly affect the agonist activity of capsaicin, resiniferatoxin (RTX) or olvanil in [Ca2+]i assays. Introducing the equivalent mutation into guinea pig TRPV1 (L549M) increased the agonist potency of PPAHV by > 10-fold in the [Ca2+]i assay and increased the amplitude of the evoked current. The rat M547L mutation reduced the affinity of RTX binding. Thus, amino acids within the S2-S4 region are important sites of agonist and antagonist interaction with TRPV1.