Polyamines Are Potent Ligands for the Capsaicin Receptor TRPV1

Polyamines are important endogenous regulators of ion channels and are known to modulate inflammation and nociception. Here we investigated effects of polyamines on the capsaicin receptor TRPV1, a major ion channel expressed in nociceptive sensory afferents. Extracellular spermine, spermidine, and putrescine directly activated TRPV1 in a charge-dependent manner, both in heterologous expression systems and sensory neurons. The threshold for activation by spermine was ∼500 μm at room temperature. At lower concentrations, spermine enhanced capsaicin-evoked currents with an EC50 of ∼5 μm. Further, polyamines freely permeated TRPV1 (estimated relative permeabilities compared with Na+ were between 3 and 16), and spermine reduced the single channel conductance from 96 to 49 pS. Experiments with TRPV1 mutants identified extracellular acidic residues critical for polyamine regulation. Neutralization of aspartate 646 (D646N) abolished direct activation by spermine, whereas neutralization of this same aspartate (D646N) or glutamate 648 (E648A) inhibited spermine-induced sensitization. These data show that polyamines, by virtue of their cationic charge, can regulate the activity of TRPV1. Extracellular polyamines are present in considerable concentrations in the gastrointestinal tract and at synapses, and these levels increase during inflammation and cancer. Therefore, polyamine regulation of TRPV1 in these tissues may be relevant to a variety of physiological and pathophysiological states. Polyamines are important endogenous regulators of ion channels and are known to modulate inflammation and nociception. Here we investigated effects of polyamines on the capsaicin receptor TRPV1, a major ion channel expressed in nociceptive sensory afferents. Extracellular spermine, spermidine, and putrescine directly activated TRPV1 in a charge-dependent manner, both in heterologous expression systems and sensory neurons. The threshold for activation by spermine was ∼500 μm at room temperature. At lower concentrations, spermine enhanced capsaicin-evoked currents with an EC50 of ∼5 μm. Further, polyamines freely permeated TRPV1 (estimated relative permeabilities compared with Na+ were between 3 and 16), and spermine reduced the single channel conductance from 96 to 49 pS. Experiments with TRPV1 mutants identified extracellular acidic residues critical for polyamine regulation. Neutralization of aspartate 646 (D646N) abolished direct activation by spermine, whereas neutralization of this same aspartate (D646N) or glutamate 648 (E648A) inhibited spermine-induced sensitization. These data show that polyamines, by virtue of their cationic charge, can regulate the activity of TRPV1. Extracellular polyamines are present in considerable concentrations in the gastrointestinal tract and at synapses, and these levels increase during inflammation and cancer. Therefore, polyamine regulation of TRPV1 in these tissues may be relevant to a variety of physiological and pathophysiological states. Polyamines are abundant organic cations essential for normal cell division and growth. At physiological pH, polyamines are positively charged and thus can interact electrostatically with negatively charged nucleic acids and proteins, including ion channels. Indeed, polyamines have a demonstrated role in ion channel regulation. Intracellular spermine and spermidine contribute to rectification of inward rectifier K+ channels (1Lopatin A.N. Makhina E.N. Nichols C.G. Nature. 1994; 372: 366-369Crossref PubMed Scopus (755) Google Scholar, 2Ficker E. Taglialatela M. Wible B.A. Henley C.M. Brown A.M. Science. 1994; 266: 1068-1072Crossref PubMed Scopus (477) Google Scholar) and glutamate receptors (3Kamboj S.K. Swanson G.T. Cull-Candy S.G. J. Physiol. 1995; 486 (Pt. 2): 297-303Crossref PubMed Scopus (321) Google Scholar, 4Bowie D. Mayer M.L. Neuron. 1995; 15: 453-462Abstract Full Text PDF PubMed Scopus (495) Google Scholar). In addition, intracellular and extracellular polyamines can, respectively, block the Transient Receptor Potential Melastatin channels, TRPM4 2The abbreviations used are: TRPM, transient receptor potential melastatin; MES, 4-morpholineethanesulfonic acid; HEK, human embryonic kidney. (5Nilius B. Prenen J. Voets T. Droogmans G. Pflugers Arch. Eur. J. Physiol. 2004; 448: 70-75Crossref PubMed Scopus (119) Google Scholar) and TRPM7 (6Kerschbaum H.H. Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 2293-2305Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Furthermore, extracellular spermine produces complex effects on NMDA (N-methyl-d-aspartate) receptors, both stimulating activity (7Rock D.M. Macdonald R.L. Mol. Pharmacol. 1992; 41: 83-88PubMed Google Scholar, 8Zhang L. Zheng X. Paupard M.C. Wang A.P. Santchi L. Friedman L.K. Zukin R.S. Bennett M.V. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10883-10887Crossref PubMed Scopus (101) Google Scholar, 9Williams K. Biochem. J. 1997; 325 (Pt. 2): 289-297Crossref PubMed Scopus (444) Google Scholar) and inducing a voltage-dependent block (10Rock D.M. MacDonald R.L. Mol. Pharmacol. 1992; 42: 157-164PubMed Google Scholar). Finally, extracellular polyamines serve as ligands for the calcium-sensing receptor (11Hofer A.M. Brown E.M. Nat. Rev. Mol. Cell Biol. 2003; 4: 530-538Crossref PubMed Scopus (541) Google Scholar), a G-protein-coupled receptor that contributes to the regulation of calcium homeostasis. Interestingly, polyamines are known to regulate inflammation and pain signaling. Levels of polyamines are raised during infection, trauma, and cancer (12Zhang M. Wang H. Tracey K.J. Crit. Care Med. 2000; 28: N60-N66Crossref PubMed Scopus (115) Google Scholar), and intrathecal administration of spermine in mice produces nocifensive behaviors (i.e. scratching, licking, and biting) characteristic of noxious stimuli (13Tan-No K. Taira A. Wako K. Niijima F. Nakagawasai O. Tadano T. Sakurada C. Sakurada T. Kisara K. Pain. 2000; 86: 55-61Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The target(s) for these effects of spermine has not been clearly defined. In this study, we investigated effects of polyamines on TRPV1, a major ion channel expressed in nociceptive primary afferent neurons. Significantly, TRPV1 is regulated by protons (14Bevan S. Geppetti P. Trends Neurosci. 1994; 17: 509-512Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 15Tominaga 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) and by extracellular cations including Na+, Mg2+, and Ca2+ (16Ahern G.P. Brooks I.M. Miyares R.L. Wang X.B. J. Neurosci. 2005; 25: 5109-5116Crossref PubMed Scopus (159) Google Scholar). These cations sensitize TRPV1 to ligand activation, and divalent cations (>5 mm) directly activate the receptor and induce pain-related behaviors in mice (16Ahern G.P. Brooks I.M. Miyares R.L. Wang X.B. J. Neurosci. 2005; 25: 5109-5116Crossref PubMed Scopus (159) Google Scholar). We therefore hypothesized that cationic polyamines could regulate TRPV1 activity. We show that extracellular spermine, spermidine, and putrescine activate TRPV1 in a charge-dependent manner. Moreover, spermine potently sensitizes TRPV1, enhancing capsaicin responses with an EC50 of ∼5 μm, a concentration that falls within the range of normal serum levels (17Milovic V. Eur. J. Gastroenterol. Hepatol. 2001; 13: 1021-1025Crossref PubMed Scopus (119) Google Scholar). These responses are abolished in TRPV1 mutants lacking key acidic residues near the extracellular pore-forming region. Thus, extracellular polyamines may represent a new class of endogenous TRPV1 ligands. All experimental procedures involving animals were approved by the Georgetown University Animal Care and Use Committee and conform to National Institutes of Health guidelines. Oocyte Electrophysiology—Xenopus laevis oocytes were injected with ∼10 ng of wild-type rat TRPV1 cRNA or mutants E600Q, D646N, and E648A (gifts from David Julius). Double electrode voltage clamp was performed using a Warner amplifier (OC725C; Warner Instruments). Oocytes were superfused (5 ml/min) with Ca2+-free solution containing (in mm) 100 NaCl, 2.5 KCl, 5 HEPES, 1 MgCl2 and titrated to pH 7.3 with ∼5 mm NaOH. For the solution containing 10 mm spermine, the concentration of NaCl was reduced to 80 mm. For solutions <pH 6.0, HEPES was replaced with 5 mm MES. Sensory Neuron and HEK293F Cell Culture and Electrophysiology—Nodose ganglia were obtained from adult mice (C57Bl6/J) and mice lacking the TRPV1 receptor (B6.129S4-Trpv1tm1Jul/J), cut, digested with collagenase, and cultured in Neurobasal +2% B-27 medium (Invitrogen), 0.1% l-glutamine, and 1% penicillin/streptomycin on poly-d-lysine-coated glass coverslips at 37 °C in 5% CO2. Neurons were used within 24-36 h of culture. HEK293F cells (Invitrogen) were cultured in Dulbecco's modified Eagle's medium supplemented with 1% non-essential amino acids and 10% fetal calf serum on poly-d-lysine-coated glass coverslips. Cells were transfected with rat TRPV1 and green fluorescent protein cDNA using Lipofectamine™ transfection reagent (Invitrogen) or Lipofectamine 2000™ (Invitrogen) according to the manufacturer's instructions. Whole-cell patch clamp and excised patch recordings were performed using an EPC8 amplifier (HEKA). The current signal was low pass filtered at 1-3 kHz and sampled at 4 kHz. The bath solution contained (in mm) 140 NaCl, 4 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, 10 glucose, pH 7.3 (290 mosm). The pipette solution contained (in mm) 140 CsCl, 10 NaCl, 10 HEPES, 5 EGTA, 2 MgATP, and 0.3 GTP, pH 7.3. For measurements of polyamine permeability the bath contained (in mm) 30 spermine, spermidine, or putrescine, 10 HEPES, pH 7.3 (titrated with HCl). Osmolarity was adjusted to 300 mosm with mannitol; the pipette solution contained (in mm) 150 NaCl, 5 EGTA, 10 HEPES, pH 7.3, buffered with NaOH. Liquid junction potentials were measured (spermine, -3 mV; spermidine, -1 mV; putrescine, +1 mV; and for comparison, 140 mm K-gluconate, +12 mV) and subtracted as previously described (18Neher E. Methods Enzymol. 1992; 207: 123-131Crossref PubMed Scopus (1008) Google Scholar). Permeability ratios were calculated as follows: PX/PNa = Na+i exp(ΔVrevF/RT)(1 + exp(ΔVrevF/RT))/z2XO, where X represents a polyamine cation of valency, z. Ion activity coefficients were 0.75 for Na+, 0.1 for spermine, 0.195 for spermidine, and 0.5 for putrescine estimated from published values for polyvalent ions at 50 mm (19Speight J.G. Lange's Handbook of Chemistry. 16th Ed. McGraw-Hill, New York2005: 299-301Google Scholar). Polyamines Activate TRPV1 in HEK293 Cells and Oocytes—We examined the effects of spermine on the function of TRPV1 expressed in HEK293 cells. Fig. 1A shows that extracellular spermine activated inward currents in voltage-clamped cells. This activation occurred in a dose-dependent manner; 500 μm and 5 mm spermine induced responses that were ∼9 and 29% of that produced by 30 nm capsaicin (Fig. 1B). Spermine activated currents in all 65 cells tested. In contrast, no responses to spermine were observed in cells transfected with green fluorescent protein alone (n = 7, data not shown). To quantify further activation by spermine, we compared the magnitude of the spermine-evoked currents to the response evoked by a saturating concentration of capsaicin (1 μm). In these experiments, spermine was included in the capsaicin solution to control for possible effects on ion permeation. This analysis revealed that 5 mm spermine activated ∼15% of the maximal TRPV1 activity (Fig. 1C). Significantly, this increased to 42% after stimulation of protein kinase C, which is known to sensitize TRPV1 to agonists (20Premkumar L.S. Ahern G.P. Nature. 2000; 408: 985-990Crossref PubMed Scopus (729) Google Scholar, 21Numazaki M. Tominaga T. Toyooka H. Tominaga M. J. Biol. Chem. 2002; 277: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 22Vellani V. Mapplebeck S. Moriondo A. Davis J.B. McNaughton P.A. J. Physiol. 2001; 534: 813-825Crossref PubMed Scopus (450) Google Scholar). We also observed spermine activation of TRPV1 in Xenopus oocytes. In this system, however, TRPV1 exhibited an apparent lower sensitivity. Under control conditions, 1 and 10 mm spermine evoked only small currents at positive potentials (Fig. 1D), but much larger responses were evident after activation of protein kinase C (Fig. 1E). Next, we tested the ability of the related polyamines, spermidine and putrescine, to activate TRPV1 in HEK293 cells. Fig. 2A shows that 5-mm concentrations of these polyamines activated TRPV1 in a charge-dependent manner; spermine (+4) was approximately twice as effective as spermidine (+3), whereas putrescine (+2) evoked little detectable current. To explore the permeability of TRPV1 to polyamines, we measured the current-voltage relationship under bionic conditions with NaCl in the pipette and with extracellular solutions containing 30 mm spermine, spermidine, or putrescine as sole charge carrier. Spermine and spermidine provoked pronounced inward currents and shifted the reversal potentials to more positive values (∼19 and 14 mV, respectively, Fig. 2C). These values correspond with a relative permeability of polyamines to Na+ (PX/PNa) of ∼15.9 and 10.5, respectively (assuming polyamine activity coefficients of 0.1 and 0.195). Putrescine alone evoked little detectable inward current but increased the current evoked by 30 nm capsaicin and produced a small negative shift in the reversal potential of -2 mV (Fig. 2D). This corresponds to a PX/PNa of ∼3.4 (using an activity coefficient of 0.5). Thus, polyamines permeate TRPV1 and the channel exhibits a high degree of selectivity toward these cations.FIGURE 2Relative activation and permeation of TRPV1 by polyamines. A, structures of the polyamines, spermine, spermidine, and putrescine. B, summary of currents produced by 5-mm concentrations of spermine, spermidine, and putrescine in HEK293 cells. Currents are expressed as a percentage of the response to spermine and were obtained from 3-4 cells for each point. C, current-voltage relationship for responses produced by 30-mm concentrations of either spermine or spermidine with the pipette solution containing 150 mm NaCl (liquid junction potentials were subtracted as described under “Materials and Methods”). Mean reversal potentials were 19.2 ± 2.8 mV (n = 5) and 14.3 ± 1.7 mV (n = 7) for spermine and spermidine, respectively. D, current-voltage relationship for responses evoked by 30 nm capsaicin (in Na+ medium) and 30 mm putrescine + capsaicin (bold line). Reversal potentials were 0 mV and -2 ± 2 mV(n = 3), respectively, for capsaicin and putrescine + capsaicin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Spermine Excites Sensory Neurons we examined the ability of spermine to regulate TRPV1 in cultured sensory neurons. to the in HEK293 spermine activated inward currents in (n = with 500 μm and 5 mm spermine, respectively, responses and of 30 nm capsaicin (Fig. and Significantly, no responses to spermine were observed in (n = data not or in cultured from mice (Fig. = Further, the currents evoked by spermine were by the TRPV1 (5 μm, Fig. = these data that spermine TRPV1 in sensory neurons. We also the effects of spermine in neurons. Spermine produced a and potentials in (Fig. = but to in cells (n = data not shown). Thus, spermine can nociceptive the activation of TRPV1, and this is effective at concentrations that not Spermine TRPV1 to in Sensory Neurons and Oocytes—We have previously that cations can sensitize TRPV1 to ligands and this with cation concentrations that are to directly the channel (16Ahern G.P. Brooks I.M. Miyares R.L. Wang X.B. J. Neurosci. 2005; 25: 5109-5116Crossref PubMed Scopus (159) Google Scholar). We therefore tested lower concentrations of spermine could capsaicin-evoked In sensory 100 μm spermine no detectable current but enhanced the currents evoked by 10 nm capsaicin (Fig. 2.8 ± = The EC50 for this was ∼5 μm (Fig. serum levels of polyamines are in the low range (17Milovic V. Eur. J. Gastroenterol. Hepatol. 2001; 13: 1021-1025Crossref PubMed Scopus (119) Google Scholar). Thus, spermine is a and relevant of TRPV1 sensitivity. This was on the capsaicin In spermine enhanced responses to a concentration of capsaicin but reduced the response to a saturating μm, Fig. At concentrations near the EC50 for capsaicin the of these was and spermine produced little in current (Fig. This with a by and T. K.J. 2005; PubMed Scopus Google Scholar) also using the expression system, that 1 mm spermine not TRPV1 currents activated by 1 μm The in current with concentrations of capsaicin a spermine-induced in single channel conductance an in Indeed, single channel recordings not revealed that spermine (5 mm) reduced the conductance from 96 ± 5 to 49 ± 5 (n = conductance at mV and and mV, Spermine at and examined the spermine regulation of TRPV1. In to effects with spermine to increase the current evoked by either protons or mm) (Fig. and These stimuli TRPV1 currents of the maximal capsaicin response (16Ahern G.P. Brooks I.M. Miyares R.L. Wang X.B. J. Neurosci. 2005; 25: 5109-5116Crossref PubMed Scopus (159) Google Scholar, Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), and therefore the of a be to a protons and divalent cations are known to regulate TRPV1 extracellular glutamate residues and (16Ahern G.P. Brooks I.M. Miyares R.L. Wang X.B. J. Neurosci. 2005; 25: 5109-5116Crossref PubMed Scopus (159) Google Scholar, Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), and therefore their ability to spermine that the may also at these To this we examined the responses to spermine in TRPV1 mutants lacking these key and We also a receptor lacking aspartate 646 (D646N) this may also contribute to regulation by P. To we measured the of capsaicin-evoked response produced by 1 mm spermine concentration we was to directly activate TRPV1 in oocytes at mV, Fig. concentrations were to the EC50 values for the TRPV1 receptor 500 500 D646N, and E600Q, Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Fig. shows that by spermine was reduced in receptors lacking either or whereas in this was enhanced To the of these residues in direct activation, we examined responses to 1 mm spermine after protein kinase C which reduced the concentration to directly activate TRPV1 Fig. 1E). We used this we observed that concentrations of spermine produced of TRPV1 currents in oocytes that this was not observed in Fig. shows that spermine-evoked currents were by of but were abolished by of In contrast, spermine evoked larger currents in mutants in wild-type In addition, spermine activated currents in these mutants in the of protein kinase C stimulation not shown). these data an important role for the acidic residues near the pore-forming and in of TRPV1 by In contrast, to little role in polyamine regulation. neutralization of this the response to This has identified polyamines as regulators of TRPV1 channel activity. We have that spermine and spermidine directly activate TRPV1 both in HEK293 cells and in sensory neurons. At room this activation occurred with concentrations however, the of TRPV1 that polyamines may be more at physiological 37 In to direct activation, we have that polyamines can sensitize TRPV1. Spermine capsaicin-evoked responses in sensory with an EC50 of ∼5 μm. In to both spermine and spermidine, putrescine to directly activate TRPV1 at concentrations as high as 30 mm but enhanced capsaicin-evoked responses (Fig. These data are with polyamines TRPV1 in a charge-dependent (spermine, This with that extracellular cations can activate and sensitize TRPV1 (16Ahern G.P. Brooks I.M. Miyares R.L. Wang X.B. J. Neurosci. 2005; 25: 5109-5116Crossref PubMed Scopus (159) Google Scholar). to divalent data that spermine with extracellular on TRPV1. by spermine was in the of high concentrations of protons or and cations are known to at extracellular glutamate residues (16Ahern G.P. Brooks I.M. Miyares R.L. Wang X.B. J. Neurosci. 2005; 25: 5109-5116Crossref PubMed Scopus (159) Google Scholar, Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), and therefore this that spermine at with TRPV1 channels the of acidic and with the alone critical for direct divalent cations or spermine not to directly interact with Interestingly, with acidic residues are also to polyamine regulation of K+ channels and glutamate receptors K. Biochem. J. 1997; 325 (Pt. 2): 289-297Crossref PubMed Scopus (444) Google Scholar). Moreover, we that polyamines, in to channel can permeate TRPV1. This to be an essential for of TRPV1 by cations block the TRPV1 can be by including Mg2+, Na+, and polyamines. the of TRPV1 at μm at μm, a K. L. K. J. V. Mol. Neurosci. 2005; PubMed Scopus Google Scholar). from block TRPV1 in a voltage-dependent T. K.J. 2005; PubMed Scopus Google Scholar). data a high selectivity of TRPV1 for polyamines, and this that TRPV1 may contribute to the of polyamines cell an polyamine is clearly in the has not been clearly identified J.G. J. Biochem. Cell Biol. 28: PubMed Scopus Google Scholar). may the of and polyamines X. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). little is known the for polyamines. In this TRPV1 and channels may represent for polyamine abundant in the intracellular considerable of polyamines may be in extracellular serum levels of polyamines are to be in the low range (17Milovic V. Eur. J. Gastroenterol. Hepatol. 2001; 13: 1021-1025Crossref PubMed Scopus (119) Google Scholar). These concentrations are of NMDA receptors (7Rock D.M. Macdonald R.L. Mol. Pharmacol. 1992; 41: 83-88PubMed Google Scholar). on the data we have levels of spermine also be to sensitize TRPV1. In addition, high concentrations of polyamines to 1 mm) may be in tissues (12Zhang M. Wang H. Tracey K.J. Crit. Care Med. 2000; 28: N60-N66Crossref PubMed Scopus (115) Google Scholar). Thus, TRPV1 could be directly activated by polyamines at these Polyamines can also in and may be from T. K. K. T. S. S. K. K. K. J. 2003; 84: PubMed Scopus Google Scholar). a could high concentrations in the to activate TRPV1, which is in the and in of In addition, and high concentrations of polyamines in the (17Milovic V. Eur. J. Gastroenterol. Hepatol. 2001; 13: 1021-1025Crossref PubMed Scopus (119) Google Scholar). These polyamines could regulate TRPV1 expressed in cells the and small or in sensory neurons. These direct or could contribute to the known effects of polyamines in the gastrointestinal tract P. O. G. Eur. J. Gastroenterol. Hepatol. 2001; 13: PubMed Scopus Google Scholar). Interestingly, spermine is in and of spermine produces pain-related behaviors in mice (13Tan-No K. Taira A. Wako K. Niijima F. Nakagawasai O. Tadano T. Sakurada C. Sakurada T. Kisara K. Pain. 2000; 86: 55-61Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). These have been to activation of NMDA receptors are inhibited by NMDA receptor that activation of TRPV1 on primary sensory contributes to the induced by of TRPV1 in this be with to NMDA TRPV1 activation of in the In we have for the that extracellular polyamines and permeate the TRPV1 ion We that polyamines may represent a new class of endogenous TRPV1 ligands. We for the and for cation activity