As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3 -carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism. TRPA1, also known as ANKTM1 and p120, belongs to the transient receptor potential (TRP) superfamily, which consists of a large group of cation channels present in species ranging from yeast to mammals (Montell et al., 2002; Clapham, 2003). In mammals, more than 20 TRP channels have been discovered, playing critical roles in physiological processes ranging from vasorelaxation, fertility, and cell growth to sensory function. Mammalian TRP channels can be divided into TRPC, TRPV, TRPM, TRPML, TRPP, and TRPA subfamilies. TRPA1 is the only member of the TRPA subfamily and is restrictively expressed in sensory neurons of dorsal root ganglia, trigeminal ganglia, and hair cells of the inner ear (Jaquemar et al., 1999; Story et al., 2003; Corey et al., 2004; Bautista et al., 2005; Nagata et al., 2005; Obata et al., 2005). In dorsal root ganglia (DRG) or trigeminal ganglia, it is specifically colocalized with TRPV1, CGRP and the bradykinin receptors. The TRPA1 channel was shown to be activated by cold stimuli with a temperature threshold of 17°C, which approximates the pain-inducing threshold of noxious cold (Story et al., 2003). TRPA1 knockout mice displayed Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.033621. ABBREVIATIONS: TRP, transient receptor potential; DRG, dorsal root ganglion; ITC, allyl isothiocyanate; URB597, 3 -carbamoylbiphenyl-3-yl cyclohexylcarbamate; FAAH, fatty acid amide hydrolase; HEK, human embryonic kidney; NGF, nerve growth factor; FLIPR, fluorometric imaging plate reader; URB532, 4-(benzyloxy)phenyl butylcarbamate; compound 7, 1-(oxazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-one; Po, open probability. 0026-895X/07/7105-1209–1216$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 5 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 33621/3203025 Mol Pharmacol 71:1209–1216, 2007 Printed in U.S.A. 1209 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from impaired sensory functions, including diminished response to environmental irritants, noxious cold, and mechanical stimuli as well as deficits in bradykinin-evoked pain hypersensitivity (Bautista et al., 2006; Kwan et al., 2006). Intrathecal injection of TRPA1-specific antisense prevented and reversed cold hyperalgesia induced by inflammation and nerve injury in pain models (Obata et al., 2005; Katsura et al., 2006). Furthermore, the sensory function of TRPA1 seems to be evolutionarily conserved. In the Drosophila melanogaster genome, two of the four counterparts of mammalian TRPA1 participate in sensory processing. dTRPA1 can be activated by warm temperatures and is required for normal thermotaxis (Viswanath et al., 2003; Rosenzweig et al., 2005). Another ortholog, painless, is required for responses to noxious heat, mechanical stimuli, and wasabi (Tracey et al., 2003; Al-Anzi et al., 2006). Together, these studies suggest that TRPA1 plays an important role in sensory function. Despite the recent interest in TRPA1, endogenous chemical ligands that activate TRPA1 remain elusive. Besides noxious cold, TRPA1 can be activated by environmental irritants such as acrolein, 2-pentenal, and various pungent natural products, including mustard oil [allyl isothiocyanate (ITC)], cinnamon oil (cinnamaldehyde), garlic (allicin), clove oil (eugenol), wintergreen oil (methyl salicylate), ginger (gingerol), and oregano (carvacrol) (Bandell et al., 2004; Jordt et al., 2004; Bautista et al., 2005; Macpherson et al., 2005; Xu et al., 2006). In general, these agents are not optimal tools for pharmacological studies. For example, ITC, cinnamaldehyde, allicin, and acrolein activate TRPA1 through covalent modification of cysteine residues within the N terminus of the channel (Hinman et al., 2006). These highly reactivate agents also have the potential to modify other proteins in a random manner. In addition, eugenol, gingerol, and icilin, which activate several other TRP channels, are not potent or efficacious on TRPA1, and mechanisms of activation by these agents are unknown. Together these factors have significantly limited application of these agents to the study of TRPA1. As part of an effort to identify novel TRPA1 agonists and antagonists, we have found that 3 -carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597) activated TRPA1 channels. URB597 was described previously as an inhibitor of fatty acid amide hydrolase (FAAH), which degrades the endogenous cannabinoid anandamide. Using Ca influx assays and patch-clamp electrophysiology, we demonstrated that URB597 activated recombinant human and rat TRPA1 channels transiently expressed in HEK293-F cells, as well as rat TRPA1 expressed in cultured DRG neurons. We also found that URB597 had an antagonist effect on TRPM8 but had no effect on TRPV1 or TRPV4 activity. Materials and Methods Transient Expression of Human and Rat TRPA1 Channels in HEK293-F Cells. Human TRPA1, TRPV1, TRPM8, TRPV4, and rat TRPA1 full-length cDNA were cloned in pcDNA3.1/V5-His Topo vector (Invitrogen, Carlsbad, CA). Transient transfections were performed using FreeStyle 293 Expression System (Invitrogen) as reported previously (Chen et al., 2007). In brief, suspension FreeStyle HEK293-F cells were transfected with TRP channel cDNA alone for Ca influx experiments, or cotransfected with green fluorescent protein for electrophysiological recordings. Cells were harvested 48 h after transfection, frozen, and stored at 85°C. Upon usage, vials were quickly thawed in a 37°C water bath and aseptically transferred into conical tubes containing Freestyle media (10 ml/vial). After spinning, medium was aspirated off, and cells were resuspended at desired densities (usually at 1,000,000 cells/ml for Ca influx and 100,000 cells/ml for electrophysiological experiments). Rat Dorsal Root Ganglion Neurons. Adult male Sprague-Dawley rats ( 8 weeks old, 250 300 g) were deeply anesthetized with CO2 and sacrificed. Lumbar (L4–L6) DRGs were isolated and incubated in 0.1% collagenase (Roche, Indianapolis, IN) containing phosphate-buffered saline for 20 min followed by 20 min in 0.1% collagenase/dispase (Sigma, St. Louis, MO) and 5 to 10 min in 0.25% trypsin (Sigma) at 37°C. After washout of enzymes, DRGs were triturated with fire-polished pipettes. Cells were plated on polyethyleniminetreated glass coverslips in a 24-well plate containing Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum, 50 nM NGF, 2 mM glutamine, and 100 U/ml penicillin–streptomycin and incubated in an atmosphere of 5% CO2 at 37°C. All experiments were conducted 24 to 48 h after plating. Ca Influx Assay. Calcium influx assay was performed using the FLIPR and calcium assay kit (Molecular Devices, Sunnyvale, CA) as reported previously (Chen et al., 2007). In brief, a day before the assay, transiently transfected cells were seeded in poly(D-lysine)coated, clear-bottomed, black-walled 96-well plates and incubated overnight at 37°C. Hanks’ balanced salt solution/20 mM HEPES (Invitrogen) was used as an assay buffer. After incubation with 100 l of 1 Ca dye for 2 h at room temperature, a two-addition protocol was used for evaluating agonist activities (i.e., activation of Ca influx) and antagonist activities (i.e., inhibition of responses induced by a known agonist). To determine activation or inhibition, the following sequence was observed: 10-s baseline readout, 50 l of assay buffer or antagonist as first addition, 3 4-min readout, agonists (4 stock) as second addition, and readout for 2.5 min. Fluorescence measurement was taken every 1 s. Minimum and maximum signals were obtained before the second addition and at the end of the experiment. Whole-Cell and Inside-out Single Channel Recordings. Patch-clamp recordings in the whole-cell or inside-out configurations were carried out using an Axopatch 200B amplifier (Molecular Devices). Transfected cells or DRG neurons were seeded on cover-slips and used within 2 to 48 h. For whole-cell recordings, extracellular recording solution contained 155 mM NaCl, 5 mM KCl, 1.6 mM MgCl2, 10 mM HEPES, 12 mM dextrose and 5 mM EGTA (320 mOsm, pH adjusted to 7.4 with NaOH). The intracellular solution contained 122.5 mM potassium aspartate, 20 mM KCl, 5 mM HEPES, 1 mM MgCl2, 10 mM EGTA and 2 mM ATP-Mg (pH 7.25, 280 mOsm). Currents were elicited from a holding potential of 60 mV, or a 200-ms voltage ramp ranging from 80 to 80 mV applied every second. Data were sampled at 2 KHz, filtered at 1 KHz, and analyzed using pClamp software (version 9; Molecular Devices). For inside-out patch recordings, a single solution for both bath and pipette contained 140 mM NaCl, 2 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (300 mOsm, pH 7.4). Data were sampled at 20 KHz and filtered at 2 KHz. Events were detected using the half-threshold criterion. Rapid drug application was achieved by using a ValveLink system (AutoMate Scientific, San Francisco, CA). Reagents. URB597 and URB532 were obtained from Calbiochem (San Diego, CA). 1-(oxazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-one (compound 7) was synthesized at Abbott Laboratories. ITC, menthol, and capsaicin were obtained from Sigma-Aldrich (St. Louis, MO). Icilin was obtained from Tocris Bioscience (Ellisville, MO). Compounds were dissolved in dimethyl sulfoxide and diluted to the required concentration in assay solutions. The final dimethyl sulfoxide concentration did not exceed 0.2%, and the solvent effects were negligible. Data Analysis. Data were analyzed with FLIPR 384 or pClamp 9 (Molecular Device Corp.); concentration dose responses were derived by using Origin 7 software (OriginLab Corp., Northampton, MA). 1210 Niforatos et al. at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from Data are reported as mean S.E.M. (n indicates the number of experiments), and Student’s t test was used to test for statistical significance between groups.