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2-Aminoethoxydiphenyl Borate Is a Common Activator of TRPV1, TRPV2, and TRPV3

2004; Elsevier BV; Volume: 279; Issue: 34 Linguagem: Inglês

10.1074/jbc.m404164200

ISSN

1083-351X

Autores

Hongzhen Hu, Qihai Gu, Chunbo Wang, Craig K. Colton, Jisen Tang, Mariko Kinoshita-Kawada, Lu‐Yuan Lee, Jackie D. Wood, Michael X. Zhu,

Tópico(s)

Herbal Medicine Research Studies

Resumo

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation. The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation. The transient receptor potential (TRP) 1The abbreviations used are: TRP, transient receptor potential; 2APB, 2-aminoethoxydiphenyl borate; 4αPDD; 4α-phorbol 12,13-didecanoate; DMEM, Dulbecco's minimal essential medium; DRG, dorsal root ganglia; ECS, extracellular solution; OAG, 1-oleolyl-2-acetyl-sn-glycerol; TRPV, vanilloid subfamily of TRP; TRPC, canonical subfamily of TRP; TRPM, melastatin subfamily of TRP; MES, 2-(N-morpholino)-ethanesulfonic acid. superfamily of cation channels consists of a large number of recently identified molecules that share sequence homology with the Drosophila protein named after a phototransduction mutant called trp. According to sequence similarities, the TRP channels are further divided into subfamilies, such as TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) (see reviews in Refs. 1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (982) Google Scholar and 2Montell C. Birnbaumer L. Flockerzi V. 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Specific ligands have been found for TRPC3, TRPC6, TRPC7, TRPV1, TRPV4, TRPM2, TRPM4, TRPM5, TRPM7, and TRPM8. These include endogenous substances, such as lipids (diacylglycerol (12Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1285) Google Scholar), anandamide (13Smart D. Gunthorpe M.J. Jerman J.C. Nasir S. Gray J. Muir A.I. Chambers J.K. Randall A.D. Davis J.B. Br. J. Pharmacol. 2000; 129: 227-230Crossref PubMed Scopus (687) Google Scholar, 14Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (823) Google Scholar), and phosphatidylinositol 4,5-bisphosphate (15Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (466) Google Scholar)), nucleotides (ADP-ribose (16Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (772) Google Scholar)), and calcium ions (17Launay P. Fleig A. Perraud A.L. Scharenberg A.M. Penner R. Kinet J.P. Cell. 2002; 109: 397-407Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 18Hofmann T. Chubanov V. Gudermann T. Montell C. Curr. Biol. 2003; 13: 1153-1158Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), as well as exogenous materials either synthesized or extracted from plants, e.g. capsaicin (7Caterina M. Schumacher M. Tominaga M. Rosen T. Levine J. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7310) Google Scholar), menthol (19McKemy D.D. Neuhausser W.M. Julius D. Nature. 2002; 416: 52-58Crossref PubMed Scopus (2066) Google Scholar), and 4α-phorbol 12,13-didecanoate (4αPDD) (20Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Although several blockers are also available, except for a limited number of TRPV1 antagonists (7Caterina M. Schumacher M. Tominaga M. Rosen T. Levine J. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7310) Google Scholar, 21Tominaga M. Caterina M.J. Malmberg A.B. Rosen T.A. Gilbert H. Skinner K. Raumann B.E. Basbaum A.I. Julius D. Neuron. 1998; 21: 531-543Abstract Full Text Full Text PDF PubMed Scopus (2644) Google Scholar), other TRP inhibitors are nonspecific. Ruthenium red blocks all TRPV channels (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (982) Google Scholar, 22Clapham D.E. Montell C. Schultz G. Julius D. Pharmacol. Rev. 2003; 55: 591-596Crossref PubMed Scopus (215) Google Scholar). 2-Aminoethoxydiphenyl borate (2APB) appears to block a number of TRPC and TRPM channels (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (982) Google Scholar, 22Clapham D.E. Montell C. Schultz G. Julius D. Pharmacol. Rev. 2003; 55: 591-596Crossref PubMed Scopus (215) Google Scholar). First reported as a membrane-permeable inhibitor of inositol 1,4,5-trisphosphate receptors (23Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. (Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (786) Google Scholar, 24Ma H.T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar), 2APB was soon found to directly block native store-operated channels (25Dobrydneva Y. Blackmore P. Mol. Pharmacol. 2001; 60: 541-552PubMed Google Scholar, 26Prakriya M. Lewis R.S. J. Physiol. 2001; 536: 3-19Crossref PubMed Scopus (437) Google Scholar, 27Bootman M.D. Collins T.J. Mackenzie L. Roderick H.L. Berridge M.J. Peppiatt C.M. FASEB J. 2002; 16: 1145-1150Crossref PubMed Scopus (618) Google Scholar), sarco/endoplasmic reticulum Ca2+-ATPase pumps (28Bilmen J.G. Wootton L.L. Godfrey R.E. Smart O.S. Michelangeli F. Eur. J. Biochem. 2002; 269: 3678-3687Crossref PubMed Scopus (98) Google Scholar), mitochondrial permeability transition pore (29Chinopoulos C. Starkov A.A. Fiskum G. J. Biol. Chem. 2003; 278: 27382-27389Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and a few other ion channels (30Lemonnier L. Prevarskaya N. Mazurier J. Shuba Y. Skryma R. FEBS Lett. 2004; 556: 121-126Crossref PubMed Scopus (28) Google Scholar). The mechanism of action for 2APB is likely to be complex. In addition to inhibition, low concentrations of 2APB enhanced the activity of store-operated channels (26Prakriya M. Lewis R.S. J. Physiol. 2001; 536: 3-19Crossref PubMed Scopus (437) Google Scholar). At greater than 50 μm, 2APB activated a Ca2+-permeable nonselective cation channel with a 50-picosiemens single channel conductance and very low open probability in rat basophilic leukemia cells (31Braun F.J. Aziz O. Putney Jr., J.W. Mol. Pharmacol. 2003; 63: 1304-1311Crossref PubMed Scopus (47) Google Scholar). 2APB has been perceived as a general inhibitor of TRP channels (1Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (982) Google Scholar). However, except for TRPC3 (24Ma H.T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (534) Google Scholar, 32Trebak M. Bird G.S. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), the effects of this drug were often examined on presumptive endogenous TRP channels (33Hermosura M.C. Monteilh-Zoller M.K. Scharenberg A.M. Penner R. Fleig A. J. Physiol. 2002; 539: 445-458Crossref PubMed Scopus (166) Google Scholar, 34Prakriya M. Lewis R.S. J. Gen. Physiol. 2002; 119: 487-507Crossref PubMed Scopus (269) Google Scholar, 35Tsuzuki K. Xing H. Ling J. Gu J.G. J. Neurosci. 2004; 24: 762-771Crossref PubMed Scopus (109) Google Scholar), of which the molecular compositions are uncertain. It is possible that an unknown subunit confers the 2APB sensitivity of the native channels. Therefore, it is important to confirm the effects of 2APB on heterologously expressed TRP channels. Here, we used TRPC6, TRPM8, and TRPV1 to represent each of the three major TRP subfamilies and examined the effects of 2APB on their activities. We confirmed that TRPC6 and TRPM8 were inhibited by 2APB. However, to our surprise, 2APB activated TRPV1 expressed in HEK293 cells and in Xenopus oocytes. In rat dorsal root ganglion (DRG) neurons, 2APB elicited currents that were potentiated by capsaicin or low pH. Finally, we showed that 2APB also activated TRPV2 and V3 and therefore is a common activator of three TRPV channels. DNA Constructs, Cell Culture, and Transfections—cDNA for murine TRPC6 was cloned as previously described (36Boulay G. Zhu X. Peyton M. Jiang M. Hurst R. Stefani E. Birnbaumer L. J. Biol. Chem. 1997; 272: 29672-29680Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). cDNAs for murine TRPV1 (GenBank™ accession number AY452083) and TRPM8, murine TRPV3, and human TRPV4 were isolated from total RNA prepared from mouse DRG, mouse skin, and human endothelial cells, respectively, by reverse transcriptase-PCR using oligonucleotide primers designed based on published sequences. The correctness of the cDNAs was confirmed by DNA sequencing. cDNAs for rat TRPV1, murine TRPV2, and rat TRPV5 and TRPV6 were kindly provided by Drs. M. Caterina, M. Kanzaki, and J. Peng. For expression in HEK293 cells and intracellular Ca2+ measurements, the cDNAs were subcloned in pcDNA3. HEK293 cells were grown in Dulbecco's minimal essential medium (DMEM) containing 4.5 mg/ml glucose, 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. For intracellular Ca2+ measurements, the cells were transfected with the desired DNA constructs in the wells of 96-well plates without preseeding using LipofectAMINE 2000 (Invitrogen) following the protocol provided by the manufacturer. To prevent cell loss from subsequent washing, the wells were treated with 20 μg/ml polyornithine (molecular weight >30,000; Sigma) for >15 min and rinsed once with Hanks' balanced salt solution without Mg2+ and Ca2+. For each well, the plasmid DNA (25 ng) and LipofectAMINE 2000 (0.4 μl) were mixed in 50 μl of OptiMEM (Invitrogen) and added to the well before the addition of 120,000 cells suspended in 100 μl of the medium without antibiotics. The cells were incubated for 24–28 h without medium change. The transfection efficiency was about 70% as determined using an enhanced green fluorescence protein expression vector. For whole cell recordings, murine TRPV1, V3, and M8 were subcloned in the bicistronic expression vector, pIRES2-EGFP (Clontech, Palo Alto, CA). TRPC6 was subcloned in pEGFP-N1 (Clontech) and expressed as a green fluorescent protein fusion protein. The transfections were performed in 35-mm dishes using LipofectAMINE 2000. For expression in Xenopus oocytes, murine TRPV1 and V3 were subcloned into the pAGA3 vector (GenBank™ accession number AY452085). Whole Cell Recordings of HEK293 Cells—Transfected HEK293 cells were reseeded in 35-mm dishes 1 day after the transfection. Whole cell recordings were performed in the following day. Recording pipettes were pulled from micropipette glass (World Precision Instruments Inc, Sarasota, FL) to 2–4 MΩ when filled with a pipette solution containing 140 mm CsCl, 0.6 mm MgCl2, 1 mm EGTA, 10 mm Hepes, pH 7.20, and placed in the bath solution containing 140 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm glucose, and 10 mm Hepes, pH 7.40. Isolated cells were voltage-clamped in the whole cell mode using an EPC9 (HEKA Instruments Inc, Southboro, MA) amplifier. For TRPC6, the bath solution was changed to an external solution containing 160 mm NaCl, 7.2 mmN-methyl-d-glucamine, 10 mm Hepes, pH 7.4, after the establishment of the whole cell configuration to facilitate the detection of the TRPC6 currents. Voltage commands were made from the Pulse+Pulse Fit program (version 8.53, HEKA), and the currents were recorded at 5 kHz. Voltage ramps of 100 ms to +100 mV after a brief (20 ms) step to -100 mV from holding potential of 0 mV were applied every 0.5 s. The cells were continuously perfused with the bath solution through a gravity-driven multioutlet device with the desired outlet placed about 50 μm away from the cell being recorded. Stock solutions of 2APB, N-arachidonyl dopamine, capsaicin, capsazepine, menthol, 1-oleolyl-2-acetyl-sn-glycerol (OAG), and resiniferatoxin were made in Me2SO. Ruthenium red was dissolved in water. Drugs were diluted in the appropriate external solutions to the desired final concentrations and applied to the cell through perfusion. The acidic solution used for TRPV1 contained 140 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm glucose, 10 mm MES, pH 6.50. All of the whole cell experiments were performed at the room temperature (20–24 °C). Intracellular Ca2+Measurements—Transiently transfected HEK 293 cells in 96-well plates were washed once with an extracellular solution (ECS) containing 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 10 mm glucose, 0.1% bovine serum albumin, and 15 mm Hepes, pH 7.4, and then incubated in 50 μl of ECS supplemented with 2 μm fluo4/AM and 0.05% Pluronic F-127 (both were from Molecular Probes, Eugene, OR) at 37 °C for 60 min. Probenecid (2 mm) was included in all of the solutions to prevent the leakage of fluo4 from the cells. At the end of the incubation, the cells were washed three times with ECS and placed in 80 μl of the same solution. Intracellular Ca2+ was measured using a fluid handling integrated fluorescence plate reader (Flex Station; Molecular Devices, Sunnyvale, CA). 2APB and other drugs were diluted into ECS at three times the desired final concentrations and delivered to the sample plate by the integrated robotic eight-channel pipettor at the preprogrammed time points. The fluo4 fluorescence was read at excitation of 494 nm and emission of 525 nm from the bottom of the plate at 0.67 Hz. All of the experiments were performed at 32 °C unless indicated otherwise. cRNA Synthesis and Expression in Xenopus Oocytes—TRPV1 and V3 in the pAGA3 vector were linearized using HindIII. cRNAs were synthesized using mMessage mMachine reagents and protocols obtained from Ambion (Austin, TX). The resulting cRNAs were dissolved in diethylpyrocarbonate-treated H2O. Sexually mature female Xenopus laevis of older than 2.5 years of age were purchased from Xenopus I, Inc. (Dexter, MI). The frogs were quarantined for at least 2 weeks before being used. For oocyte isolation, small pieces of ovarian lobe were dissected out from anesthetized frogs and shaken gently at 19 °C for 90 min in a solution containing 82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm Hepes, pH 7.4, and supplemented with 1 mg/ml collagenase (Worthington Biochem, Lakewood, NJ). Denuded, healthy looking oocytes of more than 1 mm in diameter were selected and injected in a volume of 50 nl/cell with a total of 5 ng of cRNA. The injected oocytes were incubated at 19 °C for 2–5 days in sterile Barth's saline (88 mm NaCl, 1 mm KCl, 0.41 mm CaCl2, 0.33 mm Ca(NO3)2, 0.82 mm MgSO4, 2.4 mm NaHCO3, 7.5 mm Tris-HCl, pH 7.6, supplemented with 20 units/ml penicillin and 20 μg/ml streptomycin). The solution was changed daily. Two-electrode Voltage Clamp—The oocytes were placed in a 50-μl chamber that was perfused with a bath solution containing 100 mm NaCl, 2.5 mm KCl, 1 mm MgCl2, 1.5 mm EGTA, 5 mm Hepes, pH 7.4. The cell was impaled with two intracellular glass electrodes filled with 3 m KCl connected to a TEV-700 two-electrode voltage clamp work station (Warner Instruments, Hamden, CT). The oocytes were clamped at -40 mV, and the currents were continuously recorded using a chart recorder (Astro Med, Inc., West Warwick, RI) and at the same time digitized at 100 Hz using a PowerLab Data Acquisition System (ADInstruments, Colorado Springs, CO). The low pH solutions were made of 100 mm NaCl, 2.5 mm KCl, 1 mm MgCl2, 1.5 mm EGTA, 5 mm MES, adjusted to the desired pH with NaOH. 2APB, capsaicin, and other drugs were dissolved in the bath solution at the desired final concentrations and applied to the cells by perfusion. Temperature changes were made using a CL-100 Bipolar temperature controller connected to a SC-20 dual in-line solution heater/cooler (Warner Instruments). Isolation and Culture of Rat DRG Neurons—Male Sprague-Dawley rats (150–220 g) were anesthetized with 4% halothane in air and decapitated. DRG (T1–T10) were extracted under a dissecting microscope and placed in ice-cold DMEM/Ham's F-12 solution. Each ganglion was desheathed, cut into ∼10 pieces, placed in 0.125% type IV collagenase, and incubated in a humidified chamber for 1 h in 5% CO2 in air at 37 °C. The ganglion suspension was centrifuged (150 × g, 5 min) and supernatant-aspirated. The ganglion pellet was resuspended in 0.05% trypsin and 0.53 mm EDTA in Hanks' balanced salt solution, incubated for 5 min, and centrifuged (150 × g, 5 min). The ganglion pellet was then resuspended in a modified DMEM/Ham's F-12 (DMEM/Ham's F-12 supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 μm MEM nonessential amino acids) and gently triturated with a small bore fire-polished Pasteur pipette. The dispersed cell suspension was centrifuged (500 × g, 8 min) through a layer of 15% bovine serum albumin to separate the cells from the myelin debris. The pellets were resuspended in the modified DMEM/Ham's F-12 solution supplemented with 50 ng/ml 2.5S nerve growth factor, plated onto poly-l-lysine-coated glass coverslips, and then incubated overnight (5% CO2 in air at 37 °C). Whole Cell Perforated Patch Clamp Recording of DRG Neurons—The coverslip containing the attached cells was centered in a small volume (0.2 ml) perfusion chamber that was perfused by gravity feed with 2APB, capsazepine, ruthenium red, or vehicle (extracellular solution) at 2 ml/min, whereas the chemical stimulants (2APB, capsaicin, and acid) were delivered by a pressure-driven perfusion system (ALA-VM8; ALA Scientific Instruments, Westbury, NY), with its tip positioned to ensure that the cell was fully within the stream of the perfusate. The extracellular solution consisted of 136 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 0.33 mm NaH2PO4, 10 mm glucose, 10 mm Hepes, pH 7.4. The intracellular solution contained 92 mm potassium gluconate, 40 mm KCl, 8 mm NaCl, 1 mm CaCl2, 0.5 mm MgCl2, 10 mm EGTA, 10 mm Hepes, pH 7.2. Recordings were made in the whole cell perforated patch configuration (100 μg/ml gramicidin) using Axopatch 200B/pCLAMP9 (Axon Instruments, Union City, CA). The experiments were performed at room temperature (20–24 °C). The data were acquired at 5 kHz and filtered at 2 kHz. Series resistance was compensated at ∼80%. The membrane potential was held at -70 mV. The data were collected from only one cell/dish to avoid possible drug contamination of the cells. Differential Effects of 2APB on TRP Channels—In transiently transfected HEK293 cells, the OAG (10 μm)-evoked TRPC6 currents, and the 100 μm menthol-elicited TRPM8 currents were inhibited by 2APB in a dose-dependent manner (Fig. 1, A and B). When activated, both channels gave rise to outwardly rectifying currents, which reversed near 0 mV. 2APB blocked both the inward and the outward currents with the blockage of the inward current being slightly more effective. The TRPC6 currents were incompletely blocked by 300 μm 2APB to 47.6 ± 6.7% at +100 mV and 35.4 ± 4.3% at -100 mV of the control response (n = 5). The IC50 values were 10.4 ± 2.5 μm at -100 mV (Hill coefficient, nh = 1.0 ± 0.3, n = 5) and 13.9 ± 2.3 μm at +100 mV (nh = 1.1 ± 0.2). The TRPM8 currents were abolished by 300 μm 2APB. The IC50 values were 7.7 ± 2.2 μm (nh = 0.9 ± 0.2, n = 5) and 11.6 ± 1.2 μm (nh = 1.2 ± 0.2) at -100 and + 100 mV, respectively. In contrast to TRPC6 and TRPM8, TRPV1 was dose-dependently activated by 2APB in the absence of any TRPV1 agonists (Fig. 1C). At 0.3–1.0 mm, 2APB evoked a current that was comparable with that elicited by 1 μm capsaicin, both in current amplitudes and the shape of the current-voltage (I/V) curves. The EC50 values of 2APB for TRPV1 activation were 197 ± 13 μm at -100 mV (nh = 1.8 ± 0.1, n = 6) and 130 ± 17 μm at +100 mV (nh = 1.5 ± 0.2). The response to 2APB was also detected in Xenopus oocytes injected with the cRNA for mouse TRPV1 (Fig. 2). Defoliculated Xenopus oocytes were injected with TRPV1 cRNA, and TRPV1 activity was measured 2–4 days later using two-electrode voltage-clamp techniques. The cells were held at -40 mV and placed in a Ca2+-free external solution to minimize the Ca2+-activated Cl- conductance and the inactivation of TRPV1 channels. Bath application of 2APB elicited inward currents only in oocytes injected with the cRNA for TRPV1 but not in uninjected oocytes or oocytes injected with the cRNA for an inactive form of TRPV1 that lacks 10 amino acids at the N terminus (GenBank™ accession number AY452084). The 2APB-evoked currents were abolished by 3 μm ruthenium red (Fig. 2, A and C). However, the competitive capsaicin antagonist, capsazepine, at 30 μm only blocked ∼30% of the response elicited by 2APB, although it almost completely inhibited the response activated by 1 μm capsaicin (Fig. 2). The weak effect of capsazepine on 2APB-induced currents was not unexpected because for the mouse TRPV1, the antagonist was also very weak at inhibiting the currents elicited by acid or heat (data not shown). Similarly poor inhibitions of acid- and heat-induced responses by capsazepine were found for rat but not human TRPV1 channels (37McIntyre P. McLatchie L.M. Chambers A. Phillips E. Clarke M. Savidge J. Toms C. Peacock M. Shah K. Winter J. Weerasakera N. Webb M. Rang H.P. Bevan S. James I.F. Br. J. Pharmacol. 2001; 132: 1084-1094Crossref PubMed Scopus (178) Google Scholar). Together, the above data demonstrate that 2APB is an activator of TRPV1. Potentiation of TRPV1 Responses by 2APB—TRPV1 is a polymodal sensor responsive to multiple stimuli such as, heat, protons, and the principal pungent ingredient of chili peppers, capsaicin (7Caterina M. Schumacher M. Tominaga M. Rosen T. Levine J. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7310) Google Scholar, 21Tominaga M. Caterina M.J. Malmberg A.B. Rosen T.A. Gilbert H. Skinner K. Raumann B.E. Basbaum A.I. Julius D. Neuron. 1998; 21: 531-543Abstract Full Text Full Text PDF PubMed Scopus (2644) Google Scholar). When expressed in HEK293 cells, TRPV1 is partially activated by low concentrations of 2APB (100 μm) or capsaicin (0.3 μm). When added together, these drugs caused a large increase in TRPV1 activity (Fig. 3A). The average increase was 9.0 ± 2.0-fold at +100 mV and 32 ± 11-fold at -100 mV (n = 7) as compared with the currents elicited by 2APB alone. Similarly, the response to 2APB was strongly potentiated by weak extracellular acidification to pH 6.5, which by itself had a very small effect on TRPV1. The average increases evoked by 2APB at pH 6.5 were 25.0 ± 6.0- and 21.0 ± 8.0-fold (n = 6) at +100 and -100 mV, respectively, as compared with the current elicited by 2APB at pH 7.4 (Fig. 3B). For the transiently transfected HEK293 cells, the response to the low concentration of 2APB as well as to that of capsaicin or protons was quite variable, presumably because of differences in the levels of channel expression. Noticeably, there was a negative association between the initial response to 2APB and the increase that was elicited by the co-application with the low level of capsaicin or weak acid. Cells that responded weakly to 2APB, which also had weak responses to capsaicin or acid, typically displayed more potentiation than those that responded strongly (Fig. 3C). The effect of 2APB on capsaicin- and acid-induced activation and that of capsaicin and acid on 2APB-evoked activation of TRPV1 were studied in more detail in Xenopus oocytes. As shown in Fig. 4A, 100 μm 2APB strongly potentiated the response induced by 0.1 and 0.3 μm capsaicin. At this 2APB concentration, the dose-response curve for capsaicin was shifted to the left with the EC50 value changed from 1.34 ± 0.12 μm (nh = 2.0 ± 0.3) in the absence of 2APB to 0.35 ± 0.08 μm (nh = 1.2 ± 0.3) in the presence of 2APB. Inversely, 0.3 μm capsaicin caused a left shift of the dose-response curve for 2APB (Fig. 4B) with a decrease in the EC50 value from 315 ± 13 μm (nh = 2.27 ± 0.24) to 34 ± 2 μm (nh = 0.92 ± 0.05). Likewise, the EC50 value for protons to activate TRPV1 changed from pH 5.220 ± 0.001 (nh = 1.37 ± 0.02) in the absence of 2APB to pH 6.036 ± 0.005 (nh = 1.00 ± 0.01) in the presence of 100 μm 2APB (Fig. 4C). The EC50 value of 2APB also decreased from 322.0 ± 0.8 μm (nh = 3.10 ± 0.04) at pH 7.5 to 159.1 ± 5.9 μm (nh = 1.60 ± 0.10) at pH 6.5 (Fig. 4D). These results indicate that 2APB and the other two activators of TRPV1 (capsaicin and acid) sensitized the effects of each other. Interestingly, with the presence of another stimulus, the Hill coefficient of the dose-response curve to a given ligand was typically decreased to half of the original value when it was applied alone, suggesting that sensitization is accompanied with a decrease in cooperativity. In addition to the chemical activators, the response to 2APB was also enhanced by heat. At 40 °C, the TRPV1 current evoked by 100 μm 2APB was about nine times larger than that obtained at 22 °C (Fig. 4E). Therefore, 2APB acts synergistically with other known activating factors of TRPV1 channels. Effect of 2APB on DRG Neurons—To examine its effects on native capsaicin receptors, we applied 2APB to acutely cultured rat DRG neurons. In capsaicin-sensitive cells held at -70 mV with perforated patches, 2APB dose-dependently evoked an inward current (Fig. 5A), which was inhibited by 3 μm ruthenium red (Fig. 5B) and, to a lesser extent, 10 μm capsazepine (Fig. 5C). Although 30 μm 2APB alone did not activate any current, it significantly increased the current induced by 0.3 μm capsaicin (Fig. 6A). Similarly, the response to weak acid (pH 6.5) was also strongly increased in the presence of 30 and 100 μm 2APB

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