Phosphorylation of Vanilloid Receptor 1 by Ca2+/Calmodulin-dependent Kinase II Regulates Its Vanilloid Binding
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m311448200
ISSN1083-351X
AutoresJooyoung Jung, Jae Soo Shin, Soon-Youl Lee, Sun Wook Hwang, Jaeyeon Koo, Hawon Cho, Uhtaek Oh,
Tópico(s)Ion channel regulation and function
ResumoVanilloid receptor 1 (VR1), a capsaicin receptor, is known to play a major role in mediating inflammatory thermal nociception. Although the physiological role and biophysical properties of VR1 are known, the mechanism of its activation by ligands is poorly understood. Here we show that VR1 must be phosphorylated by Ca2+-calmodulin dependent kinase II (CaMKII) before its activation by capsaicin. In contrast, the dephosphorylation of VR1 by calcineurin leads to a desensitization of the receptor. Moreover, point mutations in VR1 at two putative consensus sites for CaMKII failed to elicit capsaicin-sensitive currents and caused a concomitant reduction in VR1 phosphorylation in vivo. Such mutants also lost their high affinity binding with [3H]resiniferatoxin, a potent capsaicin receptor agonist. We conclude that the dynamic balance between the phosphorylation and dephosphorylation of the VR1 channel by CaMKII and calcineurin, respectively, controls the activation/desensitization states by regulating VR1 binding. Furthermore, because sensitization by protein kinase A and C converge at these sites, phosphorylation stress in the cell appears to control a wide range of excitabilities in response to various adverse stimuli. Vanilloid receptor 1 (VR1), a capsaicin receptor, is known to play a major role in mediating inflammatory thermal nociception. Although the physiological role and biophysical properties of VR1 are known, the mechanism of its activation by ligands is poorly understood. Here we show that VR1 must be phosphorylated by Ca2+-calmodulin dependent kinase II (CaMKII) before its activation by capsaicin. In contrast, the dephosphorylation of VR1 by calcineurin leads to a desensitization of the receptor. Moreover, point mutations in VR1 at two putative consensus sites for CaMKII failed to elicit capsaicin-sensitive currents and caused a concomitant reduction in VR1 phosphorylation in vivo. Such mutants also lost their high affinity binding with [3H]resiniferatoxin, a potent capsaicin receptor agonist. We conclude that the dynamic balance between the phosphorylation and dephosphorylation of the VR1 channel by CaMKII and calcineurin, respectively, controls the activation/desensitization states by regulating VR1 binding. Furthermore, because sensitization by protein kinase A and C converge at these sites, phosphorylation stress in the cell appears to control a wide range of excitabilities in response to various adverse stimuli. Vanilloid receptor 1 (VR1) 1The abbreviations used are: VR1, vanilloid receptor 1; PKC and PKA, protein kinase C and A, respectively; HEK, human embryonic kidney; PBS, phosphate-buffered saline; RTX, resiniferatoxin; CaMKII, Ca2+-calmodulin-dependent kinase II. encodes a ligand-gated, cationic channel that is activated by capsaicin, the potent pain-causing principal of hot chili peppers (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7089) Google Scholar). VR1 belongs to TRPV, a subset of TRP channel family; members of TRP channels are typified by having six transmembrane domains and ankyrin repeats at the N terminus (2Clapham D. Nature. 2003; 426: 517-524Crossref PubMed Scopus (2146) Google Scholar). As are other channel members of the TRPV family (3Gunthorpe M.J. Benham C.D. Randall A. Davis J.B. Trends Pharmacol. Sci. 2002; 23: 183-191Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar), VR1 is activated by heat and extracellular acid (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7089) Google Scholar, 4Tominaga 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 (2586) Google Scholar). In much the same way as morphine receptors, researchers have tried to identify the endogenous activators of VR1, and as a result, 12-hydroperoxytetraenoic acid, a product of 12-lipoxygenase, was identified to activate VR1, to bind VR1, and to share structural similarity with capsaicin (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 (967) Google Scholar, 6Shin J. Cho H. Hwang S.W. 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Mutational studies have localized the regions of VR1 responsible for vanilloid binding. Using a chimeric construction with avian VR1, Jordt and Julius (12Jordt S.E. Julius D. Cell. 2002; 108: 421-430Abstract Full Text Full Text PDF PubMed Scopus (759) Google Scholar) find that transmembrane domain III and its vicinity are responsible for hydrophobic interactions with vanilloids. In addition, two individual sites in the N and C termini are known to be critical for the hydrophilic binding of vanilloids (13Jung J. Lee S.-Y. Hwang S.W. Cho H. Shin J. Kang Y.-S. Kim S. Oh U. J. Biol. Chem. 2002; 277: 44448-44454Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Even though specific regions for ligand binding are now known, the gating mechanisms of VR1 facilitated by vanilloids are largely unknown. Sensory neurons become unresponsive to capsaicin after repeated or prolonged capsaicin application (14Szolcsanyi J. Wood J. Capsaicin in the Study of Pain. Academic Press, Inc., London1993: 1-26Google Scholar), because the capsaicin-activated current or Ca2+ influx in sensory neurons desensitizes after prolonged or repeated exposure to capsaicin (15Cholewinski A. Burgess G.M. Bevan S. Neuroscience. 1993; 55: 1015-1023Crossref PubMed Scopus (127) Google Scholar, 16Docherty R.J. Yeats J.C. Bevan S. Boddeke H.W. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 828-837Crossref PubMed Scopus (276) Google Scholar, 17Koplas P.A. Rosenberg R.L. Oxford G.S. J. Neurosci. 1997; 17: 3525-3537Crossref PubMed Google Scholar). Because of their desensitizing effect on sensory neurons, capsaicin or its analogs are often used as analgesics (18Watson C.P.N. Evans R.J. Watt V.R. Pain. 1988; 33: 333-400Abstract Full Text PDF PubMed Scopus (232) Google Scholar, 19Bernstein J.E. Korman N.J. Bickers D.R. Dahl M.V. Milikan L.E. J. Am. Acad. Dermatol. 1989; 21: 265-270Abstract Full Text PDF PubMed Scopus (263) Google Scholar). The desensitization of capsaicin-evoked currents is dependent on external Ca2+ and presumably upon the activity of a Ca2+-dependent phosphatase (16Docherty R.J. Yeats J.C. Bevan S. Boddeke H.W. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 828-837Crossref PubMed Scopus (276) Google Scholar, 17Koplas P.A. Rosenberg R.L. Oxford G.S. J. Neurosci. 1997; 17: 3525-3537Crossref PubMed Google Scholar). Recently, Bhave et al. (20Bhave G. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau R.W.t. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar) suggested that protein kinase A (PKA) controls tachyphylaxis, the reduced response to repeated stimuli. However, the molecular mechanism of VR1 desensitization has remained elusive. Therefore, we sought to identify the molecular events that lead to the desensitization of VR1, and during the course of this investigation we found the mode of activation of VR1 by vanilloids. Primary Cultures and Single-channel Recording—Primary cultures of sensory neurons isolated from all levels of the dorsal root ganglions of neonatal rats were prepared as previously described (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 (967) Google Scholar, 21Oh U. Hwang S.W. Kim D. J. Neurosci. 1996; 16: 1659-1667Crossref PubMed Google Scholar). A standard patch clamp technique was used to record single-channel currents in cultured sensory neurons (22Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15149) Google Scholar). Pipette and control bath solutions contained 130 mm NaCl, 2 mm MgCl2, 5 mm KCl, 2 mm Ca2+, and 10 mm NaOH/HEPES and were adjusted to pH 7.2. In the Ca2+-free condition, the bath solution contained 130 mm NaCl, 2 mm MgCl2,5mm KCl, 5 mm EGTA, and 10 mm NaOH/HEPES (pH 7.2). All recordings were made at room temperature. Borosilicate glass pipettes (World Precision Instruments, Saratoga, FL) were pulled to a tip resistance of 2 megaohms. The glass pipettes were then polished and coated with Sylgard (Dow Corning Co., Midland, MI). Junction potentials were canceled before forming gigaseals. Once gigaseals were formed, cell-attached or inside-out patch configurations were used to study single-channel currents of capsaicin-activated channels. Channel currents were recorded using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA). The output of the amplifier was filtered at 5 KHz with an 8-pole, low pass Bessel filter built into the amplifier. The filtered output was then digitized, stored on videotape, and imported to a personal computer (IBM Pentium-compatible) to analyze channel-current events. To obtain chart traces the output of the amplifier was filtered at 500 Hz (Frequency Devices, Haverhill, MA) and fed into a thermal array chart recorder (TA-240, Gould, Valley View, OH). The half-amplitude algorithm in FETCHAN (pCLAMP 8.0, Axon Instruments) was used to detect open events. Channel open probability (Po) of single channels was calculated according to the equation, I = Po·N·i, where I is the mean current over the interval, N is the number of functional channels in the patch, and i is the single-channel current (23Spruce A.E. Standen N.B. Stanfield P.R. Nature. 1985; 316: 736-738Crossref PubMed Scopus (311) Google Scholar, 24Zhainazarov A.B. Ache B.W. J. Neurosci. 1999; 19: 2929-2937Crossref PubMed Google Scholar). Channel activity was calculated as N·Po, and Po was determined only for patches that contained less than six functional capsaicin-activated channels. Site-directed Mutagenesis—VR1 cDNA-encoded fragments were amplified by reverse transcription-PCR from cells obtained from adult rat dorsal root ganglia (SuperScript II, Invitrogen). All mutant receptors were generated as previously described (13Jung J. Lee S.-Y. Hwang S.W. Cho H. Shin J. Kang Y.-S. Kim S. Oh U. J. Biol. Chem. 2002; 277: 44448-44454Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Purified full-length DNAs were subcloned into pSDTF (a generous gift from T. Snutch of British Columbia University) or into pcDNA3 (Invitrogen) for expression in the oocytes of Xenopus laevis or in human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA) and were confirmed by DNA sequencing using Sequenase 2.0 (Amersham Biosciences). Oocyte Recording—Templates were linearized with XbaI and transcribed with SP6 RNA polymerase (Megascript, Ambion, Austin, TX). X. laevis oocytes were injected with 5-10 ng of the cRNAs of VR1 or VR1 mutants in 50 nl of diethyl pyrocarbonate-treated water. 2-5 days after injection, 2-electrode voltage clamp recordings were performed (Ehold = -60 mV) to record whole cell currents. The recording chamber was perfused at 2 ml/min with a bath solution containing 96 mm NaCl, 5 mm HEPES, 2 mm KCl, 1.8 mm CaCl2, and 1 mm MgCl2 (pH 7.4) at room temperature, as previously described (13Jung J. Lee S.-Y. Hwang S.W. Cho H. Shin J. Kang Y.-S. Kim S. Oh U. J. Biol. Chem. 2002; 277: 44448-44454Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Cell Surface Biotinylation and Immunoblot—Cell surface biotinylation of oocyte membrane was performed as described (25Prescott E.D. Julius D. Science. 2003; 300: 1284-1288Crossref PubMed Scopus (480) Google Scholar). Briefly, X. laevis oocytes were injected with 5-10 ng of the cRNAs of VR1 or S502A/T704I mutant. Four days after injection oocytes were incubated with 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) in ND96 for 30 min at 17 °C. After washing with ND96 thoroughly, cells were lysed in the lysis buffer (100 mm NaCl, 20 mm Tris-Cl (pH 7.4), 1% Triton X-100, and protease inhibitors). The clear fraction was reserved for whole-cell lysate after centrifugation at 16,000 g for 2 min at 4 °C. Biotinylated membrane proteins were captured with streptavidin-agarose slurry (Sigma) for 3 h at 4 °C. Beads were washed with lysis buffer three times and subjected to SDS-PAGE. After transfer to a polyvinylidene difluoride membrane, Western blotting was performed using anti-NVR1 antiserum (diluted 1:1000) (13Jung J. Lee S.-Y. Hwang S.W. Cho H. Shin J. Kang Y.-S. Kim S. Oh U. J. Biol. Chem. 2002; 277: 44448-44454Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Proteins were visualized using peroxidase-conjugated secondary antibody (Sigma) by enhanced chemiluminescence (ECL, Amersham Biosciences). Immunohistochemistry—HEK 293T cells transfected with VR1 and its mutant S502A/T704I were seeded on cover glasses coated with poly-l-lysine. After 24 h of incubation, cells were fixed for 10 min in phosphate-buffered saline (PBS) containing 10% formaldehyde at room temperature, washed 3 times with PBS, and permeabilized with PBS containing 0.1%(v/v) Triton X-100 for 5 min at room temperature. The cells were then rinsed with 2% bovine serum albumin for 1 h to block nonspecific protein binding and incubated at 4 °C overnight with polyclonal anti-NVR1 (1:1000 dilution) antibody raised in mice. After washing the cells were incubated with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (Zymed Laboratories Inc., 1:100 dilution) for 1 h. For nuclear staining cells were incubated with propidium iodide (Sigma). Cells were viewed under a confocal laser scanning microscope (Leica TCS, Wetzlar, Germany). [3H]Resiniferatoxin (RTX) Binding Assay—HEK 293T cells transfected with wild-type or mutant VR1s were washed with PBS containing 5 mm EDTA and stored at -80 °C until assayed. The binding assay was performed in 96-well filtration plates (Millipore, Billerica, MA) as previously described (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 (323) Google Scholar). In an assay buffer (5 mm KCl, 5.8 mm NaCl, 2 mm MgCl2, 320 mm sucrose, 10 mm HEPES (pH 7.4)) containing 0.25 mg/ml bovine serum albumin (Cohn fraction V), 1 × 105 cells were loaded in triplicate into wells that had been previously washed with assay buffer and incubated with [3H]RTX (48.0 Ci/mmol, 10-1000 pm, Amersham Biosciences) in a total reaction volume of 150 μl. The microplate-containing samples were gently shaken at 37 °C for 1 h. Nonspecific binding was defined as the radioactivity of [3H]RTX in the presence of 1 μm nonradioactive RTX (Biomol). To terminate the reaction the microplates were chilled on ice, and ice-cold α1-acid glycoprotein (1 mg/well, Sigma) in 50 μl of assay buffer was added to each well to eliminate nonspecific binding (26Szallasi A. Lewin N.E. Blumberg P.M. J. Pharmacol. Exp. Ther. 1992; 262: 883-888PubMed Google Scholar). The buffer was then allowed to drain immediately. After each wash with a buffer containing 100 μg of the α1-acid glycoprotein, the plate was suction-dried and finally completely dried by oven heating. Filters of the microplates were punched into a scintillation vial, and the [3H]RTX activity was counted. Binding data were determined using the Hill equation (27Szallasi A. Goso C. Blumberg P.M. Manzini S. J. Pharmacol. Exp. Ther. 1993; 267: 728-733PubMed Google Scholar), B = (Bmax × LHn)/(KDn + LHn), where B represents the concentration of the receptor-ligand complex, Bmax is the maximal binding capacity or receptor density, LH is the concentration of the radioactive free ligand, KD is the concentration of [3H]RTX when the half of the receptors are occupied, and n is the Hill coefficient. In Vivo Phosphorylation—HEK 293T cells were transfected with the plasmids pcDNA-VR1 or pcDNA-VR1-S502A/T704I using Lipofectamine PLUS (Invitrogen) as instructed by the manufacturer. Approximately 24 h post-transfection, the HEK 293T cells were labeled for 4 h with 0.1 mCi/ml 32P in phosphate-free minimum essential medium supplemented with nonessential amino acids and sodium pyruvate. After washing with ice-cold PBS the cells were lysed with cold immunoprecipitation buffer containing 0.2 mm sodium orthovanadate, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40 in protease inhibitor mixture (Roche Applied Science). After removing large aggregates, soluble cell lysates were immunoprecipitated with polyclonal anti-VR1 antibody and protein A-agarose. Precipitates were washed 3 times with ice-cold immunoprecipitation buffer and incubated in 2× electrophoresis sample buffer for 10 min at 55 °C. Samples were then centrifuged at 4 °C for 10 min, separated by 8% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane for immunoblotting and autoradiography. The VR1-transfected cells were also pretreated with 10 μm capsaicin for 10 min or with 10 μm capsaicin and 10 μm FK-506 before immunoprecipitation to induce VR1 desensitization. Materials—[3H]RTX (48.0 Ci/mmol) was purchased from Amersham Biosciences, nonradioactive RTX was from Biomol Research Lab, and human recombinant PKCϵ was purchased from Biomol Research Lab. The catalytic subunit of PKA (isolated from bovine heart), protein-tyrosine kinase (isolated from bovine spleen), and calmodulin-dependent protein kinase II (CaMKII) (isolated from rat brain) were purchased from Sigma. Reagents used in cell culture were purchased from Invitrogen, and all other reagents were from Sigma. Calcineurin-dependent Desensitization—To determine the molecular events underlying desensitization, whole-cell and single-channel currents activated by capsaicin treatment were recorded from cultured dorsal root ganglion neurons. As shown in Fig. 1, whole-cell currents (Icap) were activated (Ehold = -60 mV) but were desensitized with a time constant (τD) of 34.7 s after prolonged (>3 min) application of 10 μm capsaicin in all 20 dorsal root ganglion cells tested (Fig. 1A). Strong tachyphylaxis, which is a reduced response to repeated applications of agonist, was also evident (Fig. 1A). Similarly, the addition of 10 μm capsaicin to a bath of the cell-attached patch readily activated single-channel currents (icap) at a holding potential of -60 mV. However, icap was not sustained and desensitized within about 2 min in all 18 patches tested in the presence of 2 mm Ca2+ in the bath with a τD of 30.6 s after capsaicin application. Channel activity (NPo) 8 s after capsaicin addition was 2.06 ± 0.31, but this reduced to 0.01 ± 0.01 3 min after the addition, which represented a 99 ± 1% (n = 17) reduction (Fig. 1B). The desensitization of icap by adding capsaicin to the bath was largely dependent on external Ca2+, because in a Ca2+-free bath solution the NPo of icap reduced only by 20.5 ± 17.1% (n = 16) 3 min after capsaicin application, with a τD of 293.2 s (Fig. 1B, inset). Furthermore, under external Ca2+-free conditions a 10 mm caffeine challenge (caffeine releases Ca2+ from intracellular Ca2+ stores) also significantly reduced icap (80 ± 9% reduction in NPo at 5 min, p < 0.001, n = 12). Because dephosphorylation of the capsaicin-activated channel by calcineurin, a Ca2+/calmodulin-dependent phosphatase, has been suggested to cause channel desensitization (16Docherty R.J. Yeats J.C. Bevan S. Boddeke H.W. Pfluegers Arch. Eur. J. Physiol. 1996; 431: 828-837Crossref PubMed Scopus (276) Google Scholar), we applied a calcineurin inhibitor, FK-506, to baths containing cell-attached patches. The addition of 1 μm FK-506 greatly reduced the desensitization of icap (n = 10) (Fig. 1, B and C). Thus, these results suggest that the capsaicin receptor is desensitized by being dephosphorylated by calcineurin. Recovery of icap after Desensitization by CaMKII—The refractoriness of the channel to capsaicin when dephosphorylated by calcineurin further indicates that phosphorylation could in turn recover the channel from desensitization. To test this hypothesis we applied various kinases and their respective cofactors to a bath containing capsaicin receptors that had been previously desensitized. As shown in Fig. 2, icap desensitized readily in cell-attached patches (Ehold = -60 mV) after 10 μm capsaicin had been added to the bath in the presence of 2 mm Ca2+. Moreover, desensitization of icap persisted as long as the inside-out patches endured (∼40 min, n = 8). The application of 2 mm ATP alone, a substrate for kinases, did not reactivate desensitized icap in the majority of patches tested (∼70%). However, in ∼30% of the patches tested, the addition of 2 mm ATP alone recovered icap from desensitization, presumably due to the presence of kinases in some patch membranes (28Wang Y.T. Yu X.M. Salter M.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1721-1725Crossref PubMed Scopus (125) Google Scholar, 29Yu X.M. Askalan R. Keil Jr., G.J. Salter M.W. Science. 1997; 275: 674-678Crossref PubMed Scopus (557) Google Scholar). Therefore, we examined the effects of applying various kinases to patches that did not show icap recovery from the desensitization induced by 2 mm ATP. As shown in Fig. 2, the addition of a catalytic subunit of PKA (0.4 μm) along with cAMP (100 μm) failed to induce the icap recovery after desensitization. Furthermore, the application of PKCϵ (4 nm), a specific isoform of PKC known to sensitize VR1 (30Cesare P. Dekker L.V. Sardini A. Parker P.J. McNaughton P.A. Neuron. 1999; 23: 617-624Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 31Numazaki M. Tominaga T. Toyooka H. Tominaga M. J. Biol. Chem. 2002; 277: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar), in combination with its cofactor, 1,2-dioctanoyl glycerol (5 μm), failed to reactivate the desensitized receptor. Moreover, treatment with another kinase, protein-tyrosine kinase (1.5 unit/ml), also failed to induce icap recovery after desensitization. However, when CaMKII (1.5 nm) was added with 10 μm calmodulin, icap recovered from the desensitization by up to ∼93% of the initial channel activity (Fig. 2, A and B). Mutations of VR1 at Its Putative CaMKII Consensus Sites—To confirm further whether VR1 phosphorylation by CaMKII is a prerequisite of channel activation we mutated VR1 at the CaMKII phosphorylation consensus sites and tested for icap in oocytes expressing mutant VR1. VR1 has five putative phosphorylation sites (i.e. Thr-370, Ser-502, Thr-704, Ser-774, and Ser-800) for "stringent" CaMKII motifs (Hyd-XRXX(T/S)-Hyd, where X represents any amino acid, and Hyd represents a hydrophobic amino acid residue (32White R.R. Kwon Y.-G. Taing M. Lawrence D.S. Edelman A.M. J. Biol. Chem. 1998; 273: 3166-3172Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) (Fig. 3A). Initially, we mutated each CaMKII consensus site. However, unexpectedly, robust current responses to 1 μm capsaicin were observed in oocytes expressing each VR1 mutant (Fig. 3, B and C), and these currents were comparable with those of wild-type VR1. This indicates that single mutations at each CaMKII phosphorylation site are insufficient to block Icap. In the case of cystic fibrosis transmembrane conductance regulator chloride channels or other enzymes, a random phosphorylation at more than two CaMKII sites is sufficient for channel activation or enzyme function (33Shenoy S. 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However, a VR1 mutant having double mutations at Ser-502 and Thr-704 (VR1-S502A/T704I) completely lost Icap (Fig. 3, B and C). Similarly, other mutants, VR1-S502A/T704D and VR1-S502D/T704D, that disabled the two phosphorylation sites also failed to respond to capsaicin (Fig. 3C). The lack of Icap in VR1-S502A/T704I was not due to a protein channel expression failure because the mutant VR1 exhibited a similar level of expression in cell surface or whole cell lysates of oocytes as that of the wild type VR1 (Fig. 3D). A high density of VR1 staining was found to be distributed throughout the cytoplasm and in the membrane of VR1-transfected HEK cells, as has been observed by others (4Tominaga 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 (2586) Google Scholar, 37Olah Z. Szabo T. Karai L. Hough C. Fields R.D. Caudle R.M. Blumberg P.M. Iadarola M.J. J. Biol. Chem. 2001; 276: 11021-11030Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) (Fig. 3D). Likewise, VR1-S502A/T704I was also found to be expressed in the membrane and in the cytosol of transfected HEK cells (Fig. 3D). Thus, these results indicate that the phosphorylation by CaMKII is required for the activation of Icap and that phosphorylation at one of the two consensus sites is sufficient and necessary for this activation. PKA is known to sensitize VR1, which augments Icap in response to repeated applications of capsaicin (38Lopshire J.C. Nicol G.D. J. Neurosci. 1998; 18: 6081-6092Crossref PubMed Google Scholar, 39Rathee P.K. Distler C. Obreja O. Neuhuber W. Wang G.K. Wang S.Y. Nau C. Kress M. J. Neurosci. 2002; 22: 4740-4745Crossref PubMed Google Scholar). VR1 has three putative phosphorylation motifs for PKA, namely, Thr-144, Thr-370, and Ser-502, of which Thr-370 and Ser-502 are common to CaMKII sites (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7089) Google Scholar). As in the case of mutation at Thr-370 or Ser-502 (Fig. 3C), mutation at Thr-144 did not affect current activation by capsaicin (data not shown). Even mutation at all of the three PKA phosphorylation sites (VR1-T144I/T370I/S502A) elicited Icap (Fig. 3, B and C). Furthermore, because of a recent report which claimed that a putative PKA site, Ser at 116, is important for controlling the desensitization of VR1 (20Bhave G. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau R.W.t. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar), we also mutated Ser-116. As reported by Bhave and co-workers (20Bhave G. Zhu W. Wang H. Brasier D.J. Oxford G.S. Gereau R.W.t. Neuron. 2002; 35: 721-731Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar), we found that mutation at Ser-116 failed to block Icap (Fig. 3B). Similarly, because Ser-502 and Ser-800, which are common to PKC and CaMKII consensus sites and are known to mediate the sensitizing effect of PKC on VR1 (31Numazaki M. Tominaga T. Toyooka H. Tominaga M. J. Biol. Chem. 2002; 277: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar), we mutated these two phosphorylation sites to see its effect on activation by capsaicin. As reported by others (31Numazaki M. Tominaga T. Toyooka H. Tominaga M. J. Biol. Chem. 2002; 277: 13375-13378Abstra
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