Title: Agonist Recognition Sites in the Cytosolic Tails of Vanilloid Receptor 1
Abstract: Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using its capsaicin-insensitive homologue, VRL1, we localized key amino acids, Arg-114 and Glu-761, in the N- and C-cytosolic tails, respectively, that determine ligand binding. Point mutations of the two key residues resulted in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [3H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids blocked capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket. Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using its capsaicin-insensitive homologue, VRL1, we localized key amino acids, Arg-114 and Glu-761, in the N- and C-cytosolic tails, respectively, that determine ligand binding. Point mutations of the two key residues resulted in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [3H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids blocked capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket. Capsaicin, the principal pungent ingredient of hot peppers, excites sensory neurons by opening an ion channel, the vanilloid receptor 1 (VR1), 1The abbreviations used are: VR1, vanilloid receptor 1; TM, transmembrane; HEK, human embryonic kidney; RTX, resiniferatoxin; PBS, phosphate-buffered saline; VRL1, vanilloid receptor-like channel 1 1The abbreviations used are: VR1, vanilloid receptor 1; TM, transmembrane; HEK, human embryonic kidney; RTX, resiniferatoxin; PBS, phosphate-buffered saline; VRL1, vanilloid receptor-like channel 1thereby causing pain. VR1 is a ligand-gated, cationic channel that is present mainly in small nociceptive sensory neurons (1Bevan S. Szolcsanyi J. Trends Pharmacol. Sci. 1990; 11: 330-333Abstract Full Text PDF PubMed Scopus (429) Google Scholar, 2Oh U. Hwang S.W. Kim D. J. Neurosci. 1996; 16: 1659-1667Crossref PubMed Google Scholar, 3Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6979) Google Scholar). The presence of VR1 in sensory neurons leads to questions concerning the existence of endogenous capsaicin-like substances, and various lipid metabolic products of lipoxygenases or anandamide have been suggested as candidates, because they activate VR1 and are structurally similar to capsaicin (4Zygmunt P.M. Petersson J. Andersson D.A. Chuang H. Sorgard M., Di Marzo V. Julius D. Hogestatt E.D. Nature. 1999; 400: 452-457Crossref PubMed Scopus (1851) Google Scholar, 5Hwang S.W. Cho H. Kwak J. Lee S.-Y. Kang C.J. Jung J. Cho S. Min K.H. Suh Y.G. Kim D. Oh U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6155-6160Crossref PubMed Scopus (964) Google Scholar). Accordingly, a role for lipoxygenase products in the activation of VR1 during inflammation was suggested (5Hwang S.W. Cho H. Kwak J. Lee S.-Y. Kang C.J. Jung J. Cho S. Min K.H. Suh Y.G. Kim D. Oh U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6155-6160Crossref PubMed Scopus (964) Google Scholar), and in fact, bradykinin, a potent pain-causing inflammatory mediator, is now known to activate VR1 via the lipoxygenase/VR1 pathway (6Shin J. Cho H. Hwang S.W. Jung J. Shin C.Y. Lee S.-Y. Kim S.H. Lee M.-G Choi Y.H. Kim J. Haber N.A. Reichling D.B. Khasar S. Levine J.D. Oh U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10150-10155Crossref PubMed Scopus (322) Google Scholar). In addition, bradykinin also has a potential to sensitize VR1 via a phospholipase C or protein kinase C pathway (7Premkumar L.S. Ahern G.P. Nature. 1999; 408: 985-990Crossref Scopus (717) Google Scholar, 8Chuang H.-H. Prescott E.D. Kong H. Shields S. Jordt S.-E. Basbaum A.I. Chao M.V. Julius D. Nature. 2001; 411: 957-962Crossref PubMed Scopus (1066) Google Scholar, 9Numazaki M. Tominaga T. Toyooka H. Tominaga M. J. Biol. Chem. 2002; 277: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). VR1 is also activated by acid or heat at over 43 °C, a threshold temperature for pain (3Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6979) Google Scholar, 10LaMotte R.H. Campbell J.N. J. Neurophysiol. 1978; 41: 509-528Crossref PubMed Scopus (329) Google Scholar, 11Meyer R.A. Campbell J.N. Science. 1981; 213: 1527-1529Crossref PubMed Scopus (173) Google Scholar, 12Tominaga 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 (2565) Google Scholar). Moreover, because ischemic or inflamed tissues become acidic, the acid activation of VR1 is a pathologically relevant event (13Caterina M.J. Julius D. Annu. Rev. Neurosci. 2001; 24: 487-517Crossref PubMed Scopus (1292) Google Scholar). More direct evidence for the pathophysiological role of VR1 in the production of inflammatory pain came from knock-out experiments. In mice lacking VR1, thermal hyperalgesia evoked by inflammation is reduced (14Caterina M.J. Leffler A. Malmberg A.B. Martin W.J. Trafton J. Petersen-Zeitz K.R. Koltzenburg M. Basbaum A.I. Julius D. Science. 2000; 288: 306-313Crossref PubMed Scopus (2882) Google Scholar, 15Davis J.B. Gray J. Gunthorpe M.J. Hatcher J.P. Davey P.T. Overend P. Harries M.H. Latcham J. Clapham C. Atkinson K. Hughes S.A. Rance K. Grau E. Harper A.J. Pugh P.L. Rogers D.C. Bingham S. Randall A. Sheardown S.A. Nature. 2000; 405: 183-187Crossref PubMed Scopus (1476) Google Scholar). Furthermore, hyperalgesia induced by the key inflammatory mediators, bradykinin and nerve growth factor, is reduced in mice lacking VR1 (8Chuang H.-H. Prescott E.D. Kong H. Shields S. Jordt S.-E. Basbaum A.I. Chao M.V. Julius D. Nature. 2001; 411: 957-962Crossref PubMed Scopus (1066) Google Scholar). Thus, VR1 is now considered a primary molecular transducer that mediates inflammatory hyperalgesia (13Caterina M.J. Julius D. Annu. Rev. Neurosci. 2001; 24: 487-517Crossref PubMed Scopus (1292) Google Scholar). The putative topology of VR1 indicates that it belongs to a class of transient receptor potential channels possessing six transmembrane domains and two cytosolic domains at each N- and C terminus (3Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6979) Google Scholar, 16Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). VR1 appears to form a homotetramer when expressed heterologously (17Kedei N. Szabo T. Lile J.D. Treanor J.J. Olah Z. Iadarola M.J. Blumberg P.M. J. Biol. Chem. 2001; 276: 28613-28619Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). However, VR1 may form a heteromultimer with another temperature-sensitive channel, transient receptor potential V3 (18Smith G.D. Gunthorpe M.J. Kelsell R.E. Hayes P.D. Reilly P. Facer P. Wright J.E. Jerman J.C. Walhin J.-P. Ooi L. Egerton J. Charles K.J. Smart D. Randall A.D. Anand P. Davis J.B. Nature. 2002; 418: 186-190Crossref PubMed Scopus (655) Google Scholar). The cloning of VR1 resulted in the identification of many genes with sequence homology, for example, heat-sensitive vanilloid receptor-like (VRL1) channel and osmotically activated channel (VR-OAC) (19Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1236) Google Scholar, 20Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar). In addition, splicing variants of VR1, such as stretch-inhibitable channel (SIC) and VR.5′sv, were also identified (21Suzuki M. Sato J. Kutsuwada K. Ooki G. Imai M. J. Biol. Chem. 1999; 274: 6330-6335Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 22Schumacher M.A. Moff I. Sudanagunta S.P. Levine J.D. J. Biol. Chem. 2000; 275: 2756-2762Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Interestingly, none of these homologues respond to capsaicin when expressed heterologously. Capsaicin,N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide, is known to have three functional regions, i.e. a homovanillic acid, an amide bond, and an aliphatic chain, which are often referred to as the A, B, and C regions, respectively (23Walpole C.S. Wrigglesworth R. Wood J.N. Capsaicin in the Study of Pain. Academic Press, San Diego, CA1993: 63-82Google Scholar). The homovanillyl motif and amide bond regions contain dipolar groups, such as hydroxyl and carbonyl groups, which are implicated in hydrogen bonding interactions. Analyses of the structure-activity relationship of numerous capsaicin analogues suggest that these polar regions in capsaicin analogs are essential for maintaining pungency, which could be alternatively expressed as maintaining the excitation of sensory neurons (23Walpole C.S. Wrigglesworth R. Wood J.N. Capsaicin in the Study of Pain. Academic Press, San Diego, CA1993: 63-82Google Scholar, 24Szolcsanyi J. Jancso-Garbor A. Arzneim. Forsch. 1975; 25: 1877-1881PubMed Google Scholar, 25Walpole C.S. Wrigglesworth R. Bevan S. Campbell E.A. Dray A. James I.F. Masdin K.J. Perkins M.N. Winter J. J. Med. Chem. 1993; 36: 2373-2380Crossref PubMed Scopus (118) Google Scholar, 26Szallasi A. Blumberg P.M. Pharmacol. Rev. 1999; 51: 159-212PubMed Google Scholar). In contrast, the aliphatic chain in the C region, which has an optimal chain length of 8–10 carbon atoms, is presumed to interact hydrophobically with its receptors. Although a receptor model has been suggested based on the structure-activity relationships of the capsaicin analogs, structural elements in VR1 that confer specific interactions with vanilloids have not been characterized. Recently, a region spanning the third transmembrane domain (TM3) in VR1 was found to be essential for ligand binding, presumably by hydrophobic interaction with capsaicin (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). However, regions in VR1 that determine its binding to ligands, with an emphasis on hydrophilic interactions, have not been resolved. Moreover, unlike other ligand-gated channels that produce fast synaptic transmission, vanilloids are known to act on the capsaicin receptor from the intracellular side (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, 28Jung J. Hwang S.W. Kwak J. Lee S.-Y. Kang C.J. Kim W.B. Kim D. Oh U. J. Neurosci. 1999; 19: 529-538Crossref PubMed Google Scholar, 29Welch J.M. Simon S.A. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13889-13894Crossref PubMed Scopus (169) Google Scholar). This suggests that certain regions in the cytosolic tails of VR1 are targeted as ligand recognition sites. Because of the innate implication of the involvement of cytosolic tails in ligand binding, we constructed VR1/VRL1 chimeras and performed a series of deletions and site-directed mutagenesis at the N and C termini of VR1 to localize amino acid residues in each cytosolic tail that appeared critical for ligand binding. A cDNA clone encoding an open reading frame of VR1 was isolated from the dorsal root ganglion neurons of adult rats, as described previously (28Jung J. Hwang S.W. Kwak J. Lee S.-Y. Kang C.J. Kim W.B. Kim D. Oh U. J. Neurosci. 1999; 19: 529-538Crossref PubMed Google Scholar). A VRL1 cDNA was a generous gift from D. Julius of the University of California, San Francisco. The cDNA of wild-type VR1 and its various mutants were inserted into pSDTF (a generous gift from T. P. Snutch of British Columbia University) and pCDNA3 (Invitrogen) to obtain expression in oocytes and human embryonic kidney (HEK) 293T cells, respectively. VR1/VRL1 chimeras were constructed by recombinant PCR. The deletion mutants of VR1 from either N- or C-terminal cytoplasmic domains were produced by PCR. Site-directed mutagenesis was performed using a combination of two overlapping PCR fragments, which were constructed from mutagenic primers (30Higuchi R. Erlich H.A. PCR Technology: Principles and Applications for DNA Amplification. Stokton, New York1989: 61-70Crossref Google Scholar). The incorporation of engineered mutations and the fidelity of the PCR were confirmed by DNA sequencing. cDNAs were linearized with XbaI. Capped mRNAs were synthesized using an SP6 RNA polymerase in an Ambion Megascript kit, as described by the manufacturer. Expressions in oocytes of all mutants of VR1 or VR1/VRL1 chimeras were confirmed by immunoblots. VR1 and its mutants were expressed inXenopus oocytes as described previously (28Jung J. Hwang S.W. Kwak J. Lee S.-Y. Kang C.J. Kim W.B. Kim D. Oh U. J. Neurosci. 1999; 19: 529-538Crossref PubMed Google Scholar). Briefly, stage V-VI oocytes from Xenopus laevis were defolliculated with type A collagenase (Roche Molecular Biochemicals) and microinjected with 5–25 ng of the cRNAs of wild-type VR1, mutant VR1s, or VR1/VRL1 chimeras in 50 nl of diethylene pyrocarbonate-treated water. 2 to 4 days after the microinjection, capsaicin-sensitive currents were recorded using the two-electrode voltage-clamp technique. The membrane potential was held at −60 mV, and the recording solution contained the following (in mm): 96 NaCl, 5 HEPES, 2 KCl, 1.8 CaCl2, and 1 MgCl2 (pH 7.5). All experiments were performed at room temperature. Wild-type VR1 and its mutant cDNAs subcloned in pCDNA3 were transfected into HEK 293T cells using a LipofectAMINE PLUS kit (Invitrogen). 24 to 48 h after transfection, transfected cells were washed with phosphate-buffered saline (PBS), resuspended in PBS containing 5 mm EDTA and stored at −70 °C until assayed. The binding assay was carried out in 96-well filtration plates (31Acs G. Lee J. Marquez V.E. Blumberg P.M. Brain Res. Mol. Brain. Res. 1996; 35: 173-182Crossref PubMed Google Scholar) (Multiscreen separation system; Millipore) fitted with polyvinylidene difluoride membrane filters (pore size = 0.45 μm; Durapore). 1 × 105 cells were then loaded into wells that had been washed with assay buffer containing the following (in mm): 5 KCl, 5.8 NaCl, 0.75 CaCl2, 2 MgCl2, 320 sucrose, 10 HEPES (pH 7.4) containing 0.25 mg/ml bovine serum albumin (Cohn fraction V; Sigma). Cells were incubated with [3H]RTX (30–1,000 pm) in a total reaction volume of 150 μl for 1 h, with gentle shaking at 37 °C. Nonspecific binding was defined as [3H]RTX binding in the presence of 1 μm non-radioactive RTX (32Szallasi A. Blumberg P.M. Brain Res. 1990; 524: 106-111Crossref PubMed Scopus (213) Google Scholar,33Szallasi A. Blumberg P.M. Annicelli L.L. Krause J.E. Cortright D.N. Mol. Pharmacol. 1999; 56: 581-587Crossref PubMed Scopus (121) Google Scholar). After 1 h, the microplate was chilled on ice, and prechilled α1-acid glycoprotein (1 mg/well; Sigma) in 50 μl of assay buffer was added to each well to reduce nonspecific binding (33Szallasi A. Blumberg P.M. Annicelli L.L. Krause J.E. Cortright D.N. Mol. Pharmacol. 1999; 56: 581-587Crossref PubMed Scopus (121) Google Scholar,34Szallasi A. Lewin N.E. Blumberg P.M. J. Pharmacol. Exp. Ther. 1992; 262: 883-888PubMed Google Scholar). The solution in the microplates was then aspirated immediately, and the microplate filter membranes were washed with a buffer containing 100 μg of α1-acid glycoprotein, dried completely, and collected for liquid scintillation counting. The binding data were calculated using the Hill equation,B = (B max ×L Hn)/(K Dn+ L Hn), whereB represents the concentration of receptor-ligand complex,B max is the maximal binding capacity,L H is the concentration of radioactive free ligand, K D is the concentration of [3H]RTX when half of the receptors are occupied, andn is the Hill coefficient. Lysates of the oocytes injected with the cRNAs of the wild-type or mutant VR1s were obtained by centrifuging at 1,000 × g for 10 min after homogenizing the cells in PBS containing 5 mm EDTA and 1 mmphenylmethylsulfonyl fluoride. Equal amounts of whole cell lysates were separated by 12% SDS-PAGE after incubation for 10 min at 55 °C. Electroblotted membranes were incubated with antiserum raised against the N-terminal cytoplasmic domain of VR1 (NVR1) or with antiserum against the C-terminal cytoplasmic domain of VR1 (CVR1), as described previously (35Shin J.S. Wang M.-H. Hwang S.W. Cho H. Cho S.Y. Kwon M.J. Lee S.-Y. Oh U. Neurosci. Lett. 2001; 299: 135-139Crossref PubMed Scopus (31) Google Scholar). Peroxidase-conjugated goat antimouse IgG and an enhanced chemiluminescence detection kit (Amersham Biosciences) were used for the visualization. HEK 293T cells transfected with VR1 and its mutants were seeded on a cover glass coated with poly-l-lysine. After incubation for 24 h, cells were washed briefly three times with ice-cold PBS containing 0.05% bovine serum albumin and fixed for 10 min in PBS containing 10% formaldehyde at room temperature. Cells were then washed three times with PBS, permeabilized with PBS containing 0.1% (v/v) Triton X-100 for 5 min at room temperature, and rewashed. The cells were then rinsed with 2% bovine serum albumin for 1 h, to block the nonspecific binding of proteins other than antibodies, and incubated with polyclonal anti-NVR1 or -CVR1 antiserum (1:1000 dilution) at 4 °C overnight. After washing, the cells were incubated with fluorescein isothiocyanate-conjugated rabbit antimouse IgG (1:100 dilution; Zymed Laboratories Inc.) for 1 h, rewashed extensively, and incubated with propidium iodide (Sigma) for nuclear staining. Cells expressing VR1 and its mutants were viewed under a confocal laser scanning microscope (Leica TCS; Wetzlar, Germany). [3H]RTX (48.0 Ci/mmol) was purchased from Amersham Biosciences, non-radioactive RTX was fromBiomol Research Laboratories, Inc., and capsaicin and capsazepine were from Research Biochemicals, Inc. (Natick, MA). Reagents used in cell culture were purchased from Invitrogen, and all the other reagents were from Sigma, unless indicated otherwise. Data are presented as means ± S.E. Multiple comparisons of means were performed using one-way analysis of variance followed by Tukey's post hoc test. The Student'st test was used to compare two means. p values smaller than 0.05 were considered significant. The issue of the requirement of cytosolic tails for ligand binding was addressed by constructing a series of chimeras between VR1 and a capsaicin-insensitive VR1 homologue, VRL1. When 1 μm capsaicin was applied toXenopus oocytes, those expressing wild-type VR1 exhibited large inward currents (Icap) at a holding potential of −60 mV, which were blocked by 10 μmcapsazepine, a competitive antagonist of the capsaicin receptor (2Oh U. Hwang S.W. Kim D. J. Neurosci. 1996; 16: 1659-1667Crossref PubMed Google Scholar, 36Bevan S. Hothi S. Hughes G. James I.F. Rang H.P. Shah K. Walpole C.S.J. Yeats J.C. Br. J. Pharmacol. 1992; 107: 544-552Crossref PubMed Scopus (536) Google Scholar) (Fig. 1, A and B). VRL1 expressed in oocytes did not respond to capsaicin. Chimeras (chimeras c and d) constructed with the VRL1 backbone but containing either the N or C terminus of VR1, failed to exhibit Icap, suggesting the possible role of the two cytosolic regions in capsaicin activation. When both cytosolic tails of VR1 were substituted for the corresponding segments of VRL1, the chimera (chimera e) still did not respond to capsaicin. The lack of activation by capsaicin in chimera e could have been because of the lack of the VR1 functional TM3, which is implicated in hydrophobic interaction with vanilloids (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). Indeed, when the TM3 region of VR1 was added, the VR1/VRL1 chimeras (chimerasf and g) elicited Icap. In contrast, when either TM3 or the two cytosolic tails of VR1 were replaced by cognate segments of VRL1, the chimeras produced (chimerash, i, and j) failed to showIcap, even in the presence of maximal concentration (20 μm) of capsaicin. These results indicate that certain regions in the N and C termini of VR1 are required, in addition to the TM3 regions for VR1 activation by capsaicin. To localize regions in the cytosolic tails that confer capsaicin sensitivity, a series of deletion mutants of the N terminus of VR1 were constructed (Fig.2 A). When 1 μmcapsaicin was applied, oocytes expressing VR1 mutants deleted up to Asp-113, such as Δ1–35, Δ1–109, and Δ1–113, produced Icap comparable with that of wild-type VR1 (Fig. 2, B and C). In contrast, current responses were completely absent in oocytes expressing a series of VR1 deletion mutants that omitted Arg-114, namely Δ1–114, Δ1–115, Δ1–116, Δ1–117, Δ1–118, Δ1–119, Δ1–151, Δ1–199, and Δ1–432. Moreover, the insensitivity to capsaicin of the VR1 deletion mutants lacking Arg-114 was not because of the lack of mutant expression in oocytes. All mutants were found to be expressed properly to the same extent as wild-type VR1 when probed by immunoblots with a mouse antiserum raised against a segment of the C terminus, VR1 (684–838) (Fig. 2 D). When transfected to HEK 293T cells, the Δ1–114 mutant showed immunoreactivity in the cell membrane and in the cytosol, an expression pattern similar to that of the wild-type VR1 (Fig. 2 D), and which, in part, indicates membrane targeting. These results indicate that the region close to Arg-114 is essential for channel activation by capsaicin. Although Arg-114 and the vicinity of Arg-114 in the N terminus appear to be critical for activating VR1, studies on VR1/VRL1 chimeras (particularly chimera i in Fig. 1) indicate that a certain region in the C terminus of VR1 is also implicated in activation by vanilloids. Thus, we constructed a series of deletion mutants that lacked portions of the peptide sequence in the C terminus (Fig.3 A). When expressed in oocytes, deletion mutants up to Asp-765, such as Δ823–838, Δ787–838, Δ766–838, and Δ765–838, elicited anIcap that was comparable with that of the wild-type VR1 (Fig. 3, B and C). When deletion was proceeded further to the vicinity of Glu-761 to produce VR1 mutants such as Δ764–838, Δ763–838, and Δ762–838, Icap was reduced significantly compared with that of the wild-type. When deletion proceeded beyond Glu-761, to produce Δ761–838, Δ732–838, and Δ684–838, Icap was undetectable (Fig. 3, B and C). Moreover, mutants Δ761–838and Δ762–838 were found to be expressed properly at ∼85 kDa, at levels comparable with that of the wild-type when immunostained with polyclonal antibody raised against a protein segment in the N terminus, VR1 (1–432) (Fig. 3 D). Moreover, HEK 293T cells transfected with the Δ761–838 mutant showed immunoreactivity in the cell membrane and in the cytosol, which was similar to that of wild-type VR1 (Fig. 3 D). These results suggest that Glu-761 and its vicinity in the C terminus are necessary for the activation of VR1 by capsaicin. The ability of deletion mutants to bind [3H]RTX, a potent agonist of the capsaicin receptor, was examined (32Szallasi A. Blumberg P.M. Brain Res. 1990; 524: 106-111Crossref PubMed Scopus (213) Google Scholar). As shown in Fig.4 A, [3H]RTX displayed a saturable binding to HEK-VR1 cells. As reported previously (33Szallasi A. Blumberg P.M. Annicelli L.L. Krause J.E. Cortright D.N. Mol. Pharmacol. 1999; 56: 581-587Crossref PubMed Scopus (121) Google Scholar), half-maximal binding (K D) occurred at 98.0 ± 3.5 pm [3H]RTX (mean ± S.E., triple determinations, n = 3). AtK D, nonspecific binding reached ∼12% of the total binding. The Hill coefficient (n) was 1.6 ± 0.04, indicating a positive cooperativity (33Szallasi A. Blumberg P.M. Annicelli L.L. Krause J.E. Cortright D.N. Mol. Pharmacol. 1999; 56: 581-587Crossref PubMed Scopus (121) Google Scholar). Maximal specific binding (B max) was 211.2 ± 8.4 fmol/106 cells. An N terminus deletion mutant, Δ1–109, that elicitedIcap retained specific binding for [3H]RTX with a K D of 258.1 ± 8.3 pm (mean ± range, triple determinations). In contrast, cells expressing Δ1–114, which failed to showIcap, completely lost specific binding to [3H]RTX (triple determinations, n = 2; see Fig. 4 B). Similarly, deletion mutants at the C terminus, such as Δ762–838 and Δ763–838, which displayed smaller Icap, bound [3H]RTX with K D values of 382.7 ± 11.2 pm (triple preparations, n = 3) and 115.7 ± 43.0 pm (triple determinations,n = 4), respectively. In contrast, Δ761–838, which failed to elicitIcap, had no ability to specifically bind [3H]RTX (triple determinations, n = 2; see Fig. 4 C). Thus, deletion mutants that lacked one of these two regions lost current sensitivity to capsaicin and the ability to bind ligands. These results suggest that the two regions in the vicinity of Arg-114 and Glu-761 are critical for ligand binding and that the loss of these regions abrogates vanilloid activation. To determine further whether the two residues, Arg-114 and Glu-761, are required to bind vanilloids, we constructed VR1 mutants that lacked only the single amino acids i.e. Arg-114 (Δ114) or Glu-761 (Δ761) (Fig.5 A). Our results showed that a deletion as small as a single amino acid affects the ligand binding. As shown in Fig. 5 B, Δ114 and Δ761failed to elicit capsaicin-sensitive inward currents. Furthermore, the mutants did not show specific binding to [3H]RTX (Fig.5 C). Because VR1 is activated by acid and heat (3Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6979) Google Scholar, 12Tominaga 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 (2565) Google Scholar), these mutants were also tested for acid and heat sensitivity in transfected HEK cells. As shown in Fig. 5 D, both mutants elicited whole-cell currents when activated by heat at ∼46 °C, but both mutants failed to respond to acid (pH 5.5). HEK cells transfected with the wild-type VR1 elicited a sustained current response to acid (pH 5.5) and rapid activating and inactivating currents. Moreover, HEK cells transfected with Δ114 and Δ761 also elicited rapid activating and inactivating currents, which are presumably mediated by native acid-sensing ion channels in HEK cells, because similar acid-sensitive currents were observed in non-transfected HEK cells (37Gunthorpe M.J. Smith G.D. Davis J.B. Randall A.D. Pflugers Arch. 2001; 442: 668-674Crossref PubMed Scopus (101) Google Scholar). These results further confirm that the Arg-114 and the Glu-761 regions are critical for ligand binding. Because the Arg-114 and Glu-761 residues are charged, it is likely that these charges are necessary for vanilloid binding. We thus constructed VR1 mutants with opposite charges but similar side chain lengths at Arg-114 or Glu-761. As shown in Fig.6 A, when positively charged arginine at 114 was replaced by a neutral amino acid, alanine, the mutant (R114A) elicited an Icap comparable with that of the wild-type VR1. However, when the arginine at 114 was replaced by negatively charged glutamate (R114E), a significant reduction (96.9 ± 0.5% reduction, p < 0.0001,n = 13) in Icap was observed with no apparent specific [3H]RTX binding (Fig. 6,A and B). Because the adjacent amino acid, Arg-115, is also positively charged, we replaced it with aspartate (R115D). The R115D mutant also abolished the capsaicin-sensitive currents (p < 0.001, n = 6), indicating that charge at Arg-115 contributes equally to activation by capsaicin (data not shown). Similarly, when the negatively charged glutamate at 761 was changed to glutamine, a neutral amino acid that retains a similar structure, the mutant (E761Q) elicited a great reduction inIcap (98.0 ± 0.3% reduction,p < 0.0001, n = 10) and had no specific binding for [3H]RTX (Fig. 6, A andB). Furthermore, when the glutamate at 761 was substituted with positively charged lysine, the mutant (E761K) showed no current response to capsaicin and ability to bind [3H]RTX (Fig.6, A and B). R114E and E761K elicited current responses to heat but not to acid (pH 5.5) (Fig. 6 C). These results indicate that the negative charge of glutamate at 761 is necessary for ligand recognition and that the positive charge at Arg-114 determines ligand binding to the channel but to a lesser extent than the charge at Glu-761. VR1 is a molecular sensor present in small sensory neurons and is believed to be responsible for detecting various noxious stimuli. However, despite its physiological significance, the activation mechanisms of VR1 remain unknown. It has been reported that capsaicin and its analogs act on the intracellular side of the channel, suggesting the presence of ligand binding sites in the intracellular channel domains (28Jung J. Hwang S.W. Kwak J. Lee S.-Y. Kang C.J. Kim W.B. Kim D. Oh U. J. Neurosci. 1999; 19: 529-538Crossref PubMed Google Scholar). Consistent with this idea, the present study identified two regions near Arg-114 and Glu-761 in the cytosolic tails of VR1 that determine ligand binding. These areas seemed to be minimal marginal sites required for channel activation, because truncation or point mutation of these regions causes a loss of capsaicin sensitivity and concomitant loss of [3H]RTX binding capacity. Charges carried by the amino acids in the two regions appear to be minimal determinants of binding, because the introduction of neutral or oppositely charged residues at these positions causes a substantial to complete loss of ligand binding capacity. Based on the structure-activity relationships of synthetic capsaicin analogues (24Szolcsanyi J. Jancso-Garbor A. Arzneim. Forsch. 1975; 25: 1877-1881PubMed Google Scholar,25Walpole C.S. Wrigglesworth R. Bevan S. Campbell E.A. Dray A. James I.F. Masdin K.J. Perkins M.N. Winter J. J. Med. Chem. 1993; 36: 2373-2380Crossref PubMed Scopus (118) Google Scholar), it appears that the dipolar properties of phenolic hydroxide and of the amide group in capsaicin are important for its pharmacologic effects. In accordance with this proposition, it is highly conceivable that the charged residues at Glu-761, and at Arg-114 to a lesser extent, provide a hydrophilic interaction with capsaicin. Thus, in addition to the TM3 region in VR1, which is implicated in the hydrophobic interaction (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar), the two areas in the cytosolic tails also constitute a part of the capsaicin binding pocket, as has been proposed by others (26Szallasi A. Blumberg P.M. Pharmacol. Rev. 1999; 51: 159-212PubMed Google Scholar). Although the present study suggests that regions near the Arg-114 and Glu-761 residues in VR1 are key determinants of agonist binding, we cannot rule out the possibility that other regions of VR1 are also required to form a functional ligand binding pocket. Indeed, Jordt and Julius (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar) found that the TM3 region is also necessary for ligand binding, and this was confirmed by the present study (Fig. 1). Because the transmembrane domain region is embedded in a hydrophobic environment, the transmembrane region is believed to imply a hydrophobic interaction with VR1 (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). Furthermore, mutations of the sixth transmembrane domain were found to reduce [3H]RTX binding affinity (38Kuzhikandathil E.V. Wang H. Szabo T. Morozova N. Blumberg P.M. Oxford G.S. J. Neurosci. 2001; 21: 8697-8706Crossref PubMed Google Scholar). Thus, it is conceivable that there are regions other than the two loci in the cytosolic tails or the third transmembrane domain that control VR1 ligand binding. Because we approached the sensitive areas by deleting segments of VR1 from N or C terminus, the presence of additional regions in the cytosolic tails located beyond Arg-114 and Glu-761 is also possible. More precise details of the ligand recognition sites, required for ligand binding, could be obtained by analyzing VR1 structurally. Birds are not sensitive to vanilloids and can be fed hot chili peppers (26Szallasi A. Blumberg P.M. Pharmacol. Rev. 1999; 51: 159-212PubMed Google Scholar). Consistent with this, avian VR1 is insensitive to capsaicin (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). One plausible reason for the lack of capsaicin sensitivity of chick VR1 is that chick VR1 is equipped with a TM3 region that is not functional for capsaicin binding. When the TM3 region of chick VR1 is replaced with the cognate segment of rat VR1, the chick/rat VR1 chimera shows a sensitivity to capsaicin (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). This raises a question about the presence of the putative ligand recognition sites in the cytosolic tails in chick VR1. Indeed, chick VR1 has almost the same sequences in the putative ligand recognition sites as rat VR1 (see Fig.2 A and Fig. 3 A). Therefore, chick VR1 appears to have a rudimentary backbone for vanilloid binding and elicits a rudimentary response to high doses of capsaicin (27Jordt S.-E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). However, chick VR1 lacks only the functional TM3 region among the three minimal recognition sites. In contrast, VRL1, a capsaicin-insensitive homologue of VR1 with a relatively low sequence homology, failed to show current response to capsaicin even when a large segment of the transmembrane domain of VR1, including the TM3 region, was replaced (chimeraj in Fig. 1). This is, in part, because VRL1 does not have functional ligand binding sites in the cytosolic tails. A VR1/VRL1 chimera is capsaicin-sensitive only when segments of VR1 containing the ligand binding regions in cytosolic tails and the TM3 regions are present. Thus, these results, together with those obtained using chick VR1, indicate that the putative ligand binding sites in the cytosolic tails are required for capsaicin sensitivity. The activation of ligand-gated channels by ligands often requires interactions with several separate regions in the channel complexes. For example, the binding of glutamate to theN-methyl-d-aspartate receptor requires interaction between two extracellular domains, an extracellular region preceding the M1 transmembrane segment and an extracellular loop between the M3 and M4 segments (39Laube B. Hirai H. Sturgess M. Betz H. Kuhse J. Neuron. 1997; 18: 493-503Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). In contrast, the nicotinic acetylcholine receptor forms several distinct regions in the extracellular domains of its α- and non-α-subunits for ligand binding (40Corringer P.-J., Le Novere N.L. Changeux J.-P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar). In the present study, interaction between ligands and both regions in the N- and C-cytosolic tails appears to be obligatory for agonist binding, because the deletion of either of these two regions abrogates [3H]RTX binding. The functional form of VR1 appears to be a homotetramer (17Kedei N. Szabo T. Lile J.D. Treanor J.J. Olah Z. Iadarola M.J. Blumberg P.M. J. Biol. Chem. 2001; 276: 28613-28619Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Thus, vanilloids interact with the two putative regions within or between subunits of the VR1 channel complex when it is activated by capsaicin. To determine the nature of the possible receptor ligand interaction that occurs within or between VR1 subunits, we co-expressed two VR1 mutants, Δ1–114and Δ761–838, each of which failed to respond to capsaicin when expressed alone. However, the co-expression of these two mutants failed to elicit Icap or [3H]RTX binding (data not shown). Because the two mutants contained each of the two obligatory regions, the failure of the co-expressed mutants to activate Icap suggests that vanilloids interact with the two terminal regions of one VR1 subunit. One striking feature of VR1 is its activation by heat and acid (3Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6979) Google Scholar, 12Tominaga 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 (2565) Google Scholar). Moreover, VR1 activation has clinical implications, because ischemia or inflammation causes acidosis in tissues. In the present study, VR1 mutants, such as Δ114, Δ761, R114E, or E761K, lost their ability to produce capsaicin-sensitive currents but retained heat sensitivity (see Fig. 5 D and Fig.6 C). These results indicate that the two regions in each cytosolic tail probably do not confer sensitivity to heat. Consistent with this, VRL1 is activated by heat but not by capsaicin. Therefore, activation mechanisms by heat and vanilloids appear distinct. Evidence now available suggests that the activation of VR1 by acid or capsaicin is mediated by distinct molecular mechanisms. The activation of VR1 by acid occurs only when the stimulus is applied to the extracellular surface of the channel, whereas activation by vanilloids occurs when from the intracellular side (12Tominaga 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 (2565) Google Scholar, 28Jung J. Hwang S.W. Kwak J. Lee S.-Y. Kang C.J. Kim W.B. Kim D. Oh U. J. Neurosci. 1999; 19: 529-538Crossref PubMed Google Scholar). Mutations at charged residues on the extracellular surface or at a pore region of VR1 display reduced responses to acid as compared with capsaicin (29Welch J.M. Simon S.A. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13889-13894Crossref PubMed Scopus (169) Google Scholar, 41Jordt S-E. Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8134-8139Crossref PubMed Scopus (526) Google Scholar). However, in the present study, deletion or disruption of the two sites in the cytosolic tails of VR1 dissipated capsaicin-sensitive currents and acid-gated responses (see Fig. 5 D and Fig. 6 C). Thus, unlike portions of the extracellular surface of VR1, these cytosolic regions appear to be linked functionally to the activation of VR1 by acid and by vanilloids. In summary, in this study we identified minimal marginal regions in the cytosolic tails of VR1 that control its binding to ligands. Because the charges of the two amino acids in these regions appear critical to ligand binding, it is likely that these two obligatory sites share hydrophilic interaction with vanilloids. Thus, we believe that the present study provides the molecular elements of VR1 required for ligand binding. However, we cannot exclude the possible role of the intracellular domains in controlling folding or stabilization of the channel complex. The precise nature of the molecular interactions between VR1 and vanilloids should be determined by the structural analysis of VR1.