Abstract
Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.
Highlights
Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli and proposed endogenous ligands
Transient transfections of HEK293 cells followed by immunohistochemical staining for TRPV1 protein indicated that all TRPV1 cDNAs studied in this report appeared to be expressed
In situ hybridization of rabbit dorsal root ganglia (DRG) sections with a probe generated from the oTRPV1 cDNA revealed strong labeling of cells in the DRG (Fig. 1B) with expression restricted to the small and medium diameter cell bodies consistent with that seen in other species
Summary
Rabbits were found to be resistant to the acute toxicity of capsaicin [11] and were found not to have [3H]RTX binding sites [10] These observations have provided the basis for an approach to identify key regions involved in TRPV1 binding and activation by RTX and capsaicin by cloning TRPV1 from capsaicin-sensitive and insensitive species (rat [1]; human [12]; rabbit [13, 14]; chicken [15]; and guinea pig [16]). Electrophysiological studies using membrane-impermeable analogues of capsaicin [20] and mutational analysis of extracellular loops [21, 22] have identified domains that contribute to capsaicin and proton activation, respectively These studies root ganglia; PPAHV, 12-phenylacetate 13-acetate 20-homovanillate; BCTC, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide; CHO, Chinese hamster ovary; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid; r/o, rat-rabbit chimera; h/o, human-rabbit chimera; Iodo-RTX, iodo resiniferatoxin; TM, transmembrane domain. We present a model of capsaicin and RTX bound to the TM3/4 region of rat TRPV1
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