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The C-terminal Src Inhibitory Kinase (Csk)-mediated Tyrosine Phosphorylation Is a Novel Molecular Mechanism to Limit P2X3 Receptor Function in Mouse Sensory Neurons

2009; Elsevier BV; Volume: 284; Issue: 32 Linguagem: Inglês

10.1074/jbc.m109.023051

1083-351X

Marianna D’Arco, Rashid Giniatullin, Vanessa Leone, Paolo Carloni, Nicol Birsa, S. Asha Nair, Andrea Nistri, Elsa Fabbretti,

Pharmacological Receptor Mechanisms and Effects

On sensory neurons, sensitization of P2X3 receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X3 receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X3 receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X3 receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X3 receptor function and outlines a potential new target for trigeminal pain suppression. On sensory neurons, sensitization of P2X3 receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X3 receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X3 receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X3 receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X3 receptor function and outlines a potential new target for trigeminal pain suppression. ATP-activated P2X3 receptors are expressed almost exclusively by mammalian sensory neurons to play an important role in the transduction of painful stimuli to the central nervous system (1Souslova V. Cesare P. Ding Y. Akopian A.N. Stanfa L. Suzuki R. Carpenter K. Dickenson A. Boyce S. Hill R. Nebenuis-Oosthuizen D. Smith A.J. Kidd E.J. Wood J.N. Nature. 2000; 407: 1015-1017Crossref PubMed Scopus (394) Google Scholar). Activation of P2X3 receptors by ATP released during acute and chronic pain is thought to send nociceptive signals to central pain-related networks (2Burnstock G. Nat. Rev. Drug Discov. 2008; 7: 575-590Crossref PubMed Scopus (500) Google Scholar). In view of the multitude of environmental stimuli normally reaching sensory terminals, the question then arises how inappropriate activation of P2X3 receptors is normally prevented. This process may contribute to suppression of continuous pain sensation in conjunction with central synaptic inhibition. The molecular pathways triggered by algogenic substances and responsible for modulating P2X3 receptor structure and function remain incompletely understood. This topic is of particular interest because it can provide original clues for novel approaches related to treat pain. The nerve growth factor, NGF, 2The abbreviations used are: NGFnerve growth factorα,Β-meATPα,Β-methylene-ATPAUarbitrary unitsCskC-terminal Src inhibitory kinaseFSKforskolinODGn-octyl Β-d-glucopyranosidePKCprotein kinase CTrkAtyrosine kinase receptorwtwild typePKAcAMP-dependent kinasesiRNAsmall interfering RNAHEKhuman embryonic kidney cells. is one of the most powerful endogenous substances which elicit pain and inflammation via the tyrosine kinase receptor TrkA (3Pezet S. McMahon S.B. Annu. Rev. Neurosci. 2006; 29: 507-538Crossref PubMed Scopus (691) Google Scholar). This neurotrophin stimulates an intracellular cascade that elicits PKC-dependent P2X3 receptor phosphorylation with ensuing facilitation of receptor currents. Conversely, suppression of NGF signaling powerfully down-regulates P2X3 receptor function (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar). These observations are consistent with the raised NGF levels in acute or inflammatory pain conditions (3Pezet S. McMahon S.B. Annu. Rev. Neurosci. 2006; 29: 507-538Crossref PubMed Scopus (691) Google Scholar). The molecular mechanisms underlying these effects remain, however, unclear. nerve growth factor α,Β-methylene-ATP arbitrary units C-terminal Src inhibitory kinase forskolin n-octyl Β-d-glucopyranoside protein kinase C tyrosine kinase receptor wild type cAMP-dependent kinase small interfering RNA human embryonic kidney cells. A dynamic balance between tyrosine phosphorylation and dephosphorylation is a major factor controlling the activity of many neurotransmitter receptors (5Kalia L.V. Pitcher G.M. Pelkey K.A. Salter M.W. EMBO J. 2006; 25: 4971-4982Crossref PubMed Scopus (51) Google Scholar). TrkA stimulation activates intracellular signaling including Src tyrosine kinases (6Abram C.L. Courtneidge S.A. Exp. Cell Res. 2000; 254: 1-13Crossref PubMed Scopus (342) Google Scholar) that, in neurons, are important modulators of ligand-gated receptors like nicotinic (7Charpantier E. Wiesner A. Huh K.H. Ogier R. Hoda J.C. Allaman G. Raggenbass M. Feuerbach D. Bertrand D. Fuhrer C. J. Neurosci. 2005; 25: 9836-9849Crossref PubMed Scopus (132) Google Scholar), NMDA receptors (8Liu X.J. Gingrich J.R. Vargas-Caballero M. Dong Y.N. Sengar A. Beggs S. Wang S.H. Ding H.K. Frankland P.W. Salter M.W. Nat. Med. 2008; 14: 1325-1332Crossref PubMed Scopus (186) Google Scholar), and TRPV1 receptors (9Zhang X. Huang J. McNaughton P.A. EMBO J. 2005; 24: 4211-4223Crossref PubMed Scopus (585) Google Scholar). All these receptors are involved in mediating various types of pain in the spinal cord and sensory ganglia. There is, however, no available data on the role of tyrosine phosphorylation on P2X3 receptor function. The fundamental regulator of Src signaling is the C-terminal Src kinase (Csk) that blocks it via tyrosine phosphorylation (Tyr-527, Refs. 10Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Crossref PubMed Scopus (511) Google Scholar, 11Levinson N.M. Seeliger M.A. Cole P.A. Kuriyan J. Cell. 2008; 134: 124-134Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). We explored whether tyrosine phosphorylation might regulate P2X3 receptors of sensory neurons by focusing on the P2X3 C-terminal domain Tyr-393 residue, which is included in a region with significant similarity with the Csk-phosphorylating region of Src. Our data demonstrate that Csk activation induced an increased tyrosine (Tyr-393 residue) P2X3 receptor phosphorylation with decreased receptor function, observed both in mouse trigeminal sensory neurons as well as a cell expression system. We, thus, propose that Csk-mediated P2X3 receptor inhibition is a novel mechanism to limit overactivation of P2X3 receptors. pCDNA3-P2X3 (rat sequence, NCBI accession number: CAA62594) was provided by Dr. A. North (University of Manchester, UK). pCDNA3-Csk (12Lowry W.E. Huang J. Ma Y.C. Ali S. Wang D. Williams D.M. Okada M. Cole P.A. Huang X.Y. Dev. Cell. 2002; 2: 733-744Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) was kindly provided by Dr. X. Y. Huang (Cornell University). pGEX-rat P2X3 C-terminal domain (13Bernier L.P. Ase A.R. Chevallier S. Blais D. Zhao Q. Boué-Grabot E. Logothetis D. Séguéla P. J. Neurosci. 2008; 28: 12938-12945Crossref PubMed Scopus (74) Google Scholar) was gently provided by Dr. P. Seguela (McGill University). pCDNA3-P2X3 or pGEX-P2X3 mutants were obtained using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) and the following primers: Y393A 5′-GACTCAGGGGCCGCTTCTATTGGTCACTAG-3′; Y393F 5′-GACTCAGGGGCCTTTTCTATTGGTCACTAG-3′; E384A 5′-TTCACCAGCGACGCGGCCACAGCGGAG-3′ and Q380A 5′-CAGGCCACAGCGGCGAAGCAGTCCACCGAT-3′. Correct mutagenesis was confirmed by automated DNA sequencing, while correct expression was confirmed by immunofluorescence microscopy experiments and Western blotting. Primary cultures of trigeminal ganglion neurons from C57-Black mice (12–14 days old) were cultured as described and used 24 h after plating (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar). The following substances were added to the culture medium as required: anti-NGF neutralizing antibody (6 μg/ml, 1:5000; Sigma; 4); NGF (50 ng/ml; Alomone, Jerusalem, Israel), H89 (10 μm, Sigma), forskolin (10 μm, Sigma). Inhibitors were pre-applied for 30 min to serum-starved cultures. To block tyrosine phosphatases, living cells were incubated with sodium pervanadate (50 μm Na3VO4, Sigma) for 10 min at 37 °C (9Zhang X. Huang J. McNaughton P.A. EMBO J. 2005; 24: 4211-4223Crossref PubMed Scopus (585) Google Scholar, 14Khanna S. Roy S. Park H.A. Sen C.K. J. Biol. Chem. 2007; 282: 23482-23490Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Transient transfection of pCDNA3-P2X3 receptors in HEK293T cells was carried out with calcium/phosphate method. Mock transfection was obtained with co-transfected with pEGFP plasmid (Clontech, Mountain View, CA). C-terminal P2X3 peptides (amino acid residues 346–397) were used for in vitro kinase assay. Reactions were performed using 1 μg of substrate peptide and 1 μg of recombinant kinase GST-Csk (Cell Signaling Technology, Danvers, MA) according to the manufacturer’s protocol. pGEX-P2X3 peptides were expressed in Escherichia coli BL21 cells (GE Healthcare, Uppsala Sweden) and purified with a GST SpinTrap Purification module (GE Healthcare). The quality and quantity of input target peptides were checked with SDS-PAGE and Coomassie Blue staining. Activity of the commercial recombinant Csk was determined by the Supplier (Cell Signaling) using a radiometric method in a kinase dose-dependent assay based on Csk HTScanTM kit (Cell Signaling). Trigeminal cultures were lysed in ODG buffer (2% n-octyl Β-d-glucopyranoside, 1% Nonidet P-40, 10 mm Tris, pH 7.5, 150 mm NaCl, 100 mm NaF, 20 mm orthovanadate) plus protease inhibitors mixture (Complete, Roche Applied Science). For phosphorylation studies, proteins were extracted in ODG buffer, immunopurified in TNE buffer (10 mm Tris, 150 mm NaCl, 2 mm EDTA plus 100 mm NaF, 20 mm orthovanadate, and protease inhibitors) with rabbit anti-P2X3 or anti-Csk antibodies (0.5 μg/ml, Santa Cruz Biotechnology, Santa Cruz, CA) and pull down with protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 4 h at 4 °C. For co-immunoprecipitation, proteins were extracted in the presence of 1% Triton X-100. Membrane protein biotinylation experiments were performed as previously described (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar). Total cellular membranes were purified by ultracentrifugation at 100,000 × g for 1 h at 4 °C (14Khanna S. Roy S. Park H.A. Sen C.K. J. Biol. Chem. 2007; 282: 23482-23490Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Samples were separated on 10% polyacrylamide gel and processed for Western immunoblotting using the following antibodies: anti-P2X3 (1:300; Alomone), anti-Csk (1:500, Santa Cruz Biotechnology), anti-Src-p527 (1:500; Cell Signaling), anti-phospho-tyrosine horseradish peroxidase (HRP)-conjugated (1:3500; clone Y20; Invitrogen, San Giuliano Milanese, Italy). To avoid detection of immunoglobulin heavy chains in Western blot, a mouse anti-rabbit IgG-HRP-conjugated (Jackson ImmunoResearch, Suffolk, UK) was used as secondary antibody. Quality and correct reactivity of anti-Csk, anti-Src-p527 and anti-Src-p416 antibodies were tested analyzing with Western immunoblotting HEK protein lysates after Csk overexpression cells (not shown, 15Zhang S.Q. Yang W. Kontaridis M.I. Bivona T.G. Wen G. Araki T. Luo J. Thompson J.A. Schraven B.L. Philips M.R. Neel B.G. Mol. Cell. 2004; 13: 341-355Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Loading controls were performed processing total lysates with Western immunoblotting or stripping (RestoreTM, Pierce) and successive probing. Western blot signals were detected with enhanced chemiluminescence light ECL (GE Healthcare). For quantification, band density was measured using Scion Image software. Paraformaldehyde fixed trigeminal neurons were processed with a guinea pig anti-P2X3 antibody (1:400, Neuromics, Edina, MN) and a rabbit anti-Csk antibody (1:200, Santa Cruz Biotechnology) or a rabbit anti-phospho-Src527 antibody (1:50, Cell Signaling). Immunofluorescence reactions were visualized using suitable AlexaFluor-conjugated secondary antibodies, or, for triple immunofluorescence reactions, with biotinylated antibody and streptavidin-AlexaFluor647-conjugated (1:500, Invitrogen). Cells stained with secondary antibodies only, showed no immunostaining. Membrane in vivo labeling was obtained with wheat germ agglutinin (WGA) AlexaFluor488-conjugated (1:200, Invitrogen). Specimens were observed with a confocal Leica TCS SP5 microscope (Ar-He, Ne laser). Quantitative analysis was obtained with MetaMorph software (Molecular Devices, Downingtown, PA). Data are the mean of at least five independent experiments where an average of 260 cells was analyzed. For siRNA experiments, trigeminal neurons (from 2 mouse) or HEK293 cells (5 × 104 cells/well in 12-well plate) were transfected, respectively, with mouse or human Csk siRNA SmartPools (100 nm, Dharmacon RNAi Technology, Lafayette, CO) using the DharmaFECTTM-1 transfection reagent (Dharmacon). For transfection efficiency control, cells were transfected with scramble RNA and siGLO RISC-Free siRNA (Dharmacon). Efficiency of Csk silencing was tested with Western immunoblotting and immunofluorescence. 24 h after silencing HEK cells were transfected with pEGFP or pCDNA3-P2X3 plasmids and analyzed 48 h later. Trigeminal neurons or HEK cells were recorded in whole-cell configuration as described (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar). Intracellular solution of recording pipette contained (in mm): 140 KCl, 0.5 CaCl2, 2 MgCl2, 2 Mg2ATP, 2 GTP, 10 HEPES, and 10 EGTA (pH 7.2). Cells were continuously superfused (2 ml/min rate) with physiological solution containing (in mm): 152 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.4). Responses to selective P2X3 receptor agonist α,Β-methylene-ATP (α,Β-meATP, resistant to ectoATPase hydrolysis, Sigma) were measured in terms of peak amplitude. To express agonist potency in terms of concentration producing 50% of the maximum response (EC50 values), dose-response curves for α,Β-meATP were constructed by applying different agonist doses to the same cells and fitting them with a logistic equation (Origin 6.0, Microcal, Northampton, MA). The onset of desensitization was estimated by calculating the first time constant of current decay (τfast) in the presence of agonist in accordance with our previous reports. Recovery from desensitization was assessed by paired-pulse experiments (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar). To perform the sequence alignment, 346–397 amino acid residues of C-terminal portion of the rat P2X3 receptor (CAA62594) were aligned with the C-terminal amino acid residues 478–533 of Src (Q9JJ10) involved in Csk/Src interaction. To this aim, we used ClustalW and T-COFFEE algorithms (16Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar, 17Lesk A.M. Introduction to Bioinformatics. Oxford, NY2002: 160-215Google Scholar). After this step, computational homology modeling, in silico P2X3-Csk docking and alanine scanning were performed to identify candidate residues suitable for mutagenesis within the putative Csk-docking site of the P2X3 domain. Details are provided as supplemental information. Accordingly to computational predictions, we mutated potential residues (Gln-380 and Glu-384) in P2X3 sequence that might destabilize Csk-P2X3 C-terminal complex and we explored the consequences using Csk in vitro kinase assays. Data are expressed as means ± S.E., where n indicates the number of experiments in molecular biology/immunocytochemistry or the number of investigated cells in electrophysiology. Statistical analysis was performed using the Student’s t test, the Mann-Whitney rank sum test or the ANOVA test, as appropriate. A p value of ≤0.05 was accepted as indicative of a statistically significant difference. The tyrosine kinase Src is negatively controlled by the C-terminal Src kinase, Csk, via its docking to the Src C-terminal residues 504–518 (Fig. 1; 11Levinson N.M. Seeliger M.A. Cole P.A. Kuriyan J. Cell. 2008; 134: 124-134Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 18Cowan-Jacob S.W. Fendrich G. Manley P.W. Jahnke W. Fabbro D. Liebetanz J. Meyer T. Structure. 2005; 13: 861-871Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). By exploring the C-terminal region of the P2X3 receptor, we could identify a sequence homologous to the C-terminal portion of Src specifically binding Csk (40% homology, 33.3% identical residues and 6.7% strongly similar residues, Fig. 1 and supplemental Fig. S1). In particular, the P2X3 C-terminal Tyr-393 residue, a residue specific for the P2X3 subtype and is not conserved in the other P2X family members, appeared fully aligned with the target of Csk, Src Y527. Furthermore computational modeling (19Mager P.P. Weber A. Illes P. Curr. Top. Med. Chem. 2004; 4: 1657-1705Crossref PubMed Scopus (21) Google Scholar) suggested also similar accessibility of these residues to the solvent, rendering them suitable targets to a similar kinase. These observations prompted us to examine if P2X3 Tyr-393 was a target for Csk activity. Hence, we performed a cell-free in vitro kinase assay plus a Western blotting with phosphotyrosine antibodies. Using recombinant Csk and GST-P2X3 C-terminal domain purified from E. coli, we observed P2X3 tyrosine phosphorylation (n = 5; Fig. 2A, lane 1). This signal was not detectable when the same assay was run in the presence of a P2X3 antibody recognizing the P2X3 peptide 383–397 (n = 3, Fig. 2B, lane 2) or when GST-P2X3Y393F mutant was tested as substrate (n = 5, Fig. 2B, lane 3), demonstrating thus that P2X3 Tyr-393 was a target for Csk phosphorylation in vitro. Furthermore, following our computational modeling data, we performed mutagenesis of the specific P2X3 residues Q380A and E384A within the P2X3 region compatible with putative Csk docking (residues 371–386, see Fig. 1). This approach should help to explore Csk docking to the P2X3 subunits as suggested by the in vitro kinase assay (Fig. 2C). These experiments demonstrated a significantly reduced phosphorylation of mutants Q380A and E384A (50.2 ± 6.6%; and 51 ± 8.1% respectively, taken 100% wt signal, n = 3, Fig. 2C, lanes 2 and 3) in comparison with wt (lane 1), thus, supporting the existence of a Csk docking site on the P2X3 receptor C-terminal domain. Co-expression of full-length P2X3 receptors together with Csk in HEK cells was therefore carried out to investigate functional consequences of receptor tyrosine phosphorylation. Csk and P2X3 co-transfection led to robust co-expression of both proteins (Fig. 2D, lane 2) with a significant increase in P2X3 receptor tyrosine phosphorylation (Fig. 2D, lane 2; n = 5). In accordance with the notion that PKA-mediated phosphorylation of Csk is essential to enable this kinase activity (20Abrahamsen H. Vang T. Taskén K. J. Biol. Chem. 2003; 278: 17170-17177Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), we observed that, in HEK cells, the PKA inhibitor H89 (10 μm; 30 min) prevented P2X3 tyrosine phosphorylation (10 μm; 30 min; Fig. 2D, lane 3, n = 4). It is noteworthy that basal adenylyl cyclase in these cells was sufficient to trigger activation of endogenous Csk, since H89 suppressed basal P2X3 tyrosine phosphorylation (supplemental Fig. S2A). In basal condition, a small portion of Csk and P2X3 receptor interacted at cellular level, as observed by weak P2X3/Csk co-immunoprecipitation in HEK cells (Fig. 2E, lane 3). However, the co-immunoprecipitation signal was strongly detected when using the Y393F mutant (Fig. 2E, lane 2) despite the lack of phosphorylation (see Fig. 2A, lane 2). These data suggested that Csk and P2X3 receptors are part of a complex; however, highly dependent by phosphorylation state and conformation. Whole-cell patch clamp experiments on HEK cells co-transfected with Csk and P2X3 indicated that P2X3 receptor-mediated currents evoked by the selective agonist α,Β-meATP (≥10 μm) were significantly (p = 0.02 for 100 μm, p = 0.05 for 10 μm) smaller than those recorded from cells expressing the P2X3 alone (Fig. 2G; n = 10), although this effect was not associated with a significant variation in membrane receptor expression (n = 3, p > 0.05; Fig. 2F). Such smaller receptor responses were not accompanied by changes in the agonist EC50 value (indicative of drug receptor affinity, Table 1), in the Hill coefficient (indicative of the stoichiometry of drug receptor interaction, Table 1) or in the time constant of current decay (indicative of desensitization onset, 101 ± 8 ms for control versus 126 ± 10 ms for P2X3/Csk co-expression; n = 17 and 13, respectively). Likewise, no significant changes were observed in terms of P2X3 current recovery from desensitization (tested with a double agonist pulse application, 4) in the presence (58 ± 5% of the previous response; n = 8) or in the absence of Csk (60 ± 4% of the previous response, n = 18).TABLE 1Characteristics of P2X3 receptors and its Tyr-393 mutantsReceptorEC50Hill coefficientNaN indicates the number of neurons tested for these analyses.μmP2X3 wt2.05 ± 0.471.06 ± 0.1110Wt/Csk1.74 ± 0.511.05 ± 0.1310Y393A1 ± 0.211.24 ± 0.1613Y393F1.56 ± 0.660.89 ± 0.166Y393A/Csk1.02 ± 0.251.23 ± 0.1412Wt siRNA mock2.44 ± 0.921.10 ± 0.426Wt siRNA Csk1.67 ± 0.341.21 ± 0.4210a N indicates the number of neurons tested for these analyses. Open table in a new tab Globally, our results suggest that Csk phosphorylated P2X3 receptors in cells and in vitro that was associated with decreased receptor function, when tested with high agonist concentrations, without concomitant alteration in receptor kinetic properties. Because Tyr-393 appeared to be the Csk target in the in vitro assays, we mutated this residue to explore its functional consequences with patch clamp recording from transfected HEK cells. Despite similar expression observed with immunofluorescence microscopy and membrane biotinylation assay (Fig. 3, A and B), the current amplitude evoked by α,Β-meATP (100 μm) was larger in the P2X3 Y393A and Y393F mutants than in wt receptors (see examples and average data in Fig. 3C). The agonist dose response curves for P2X3 mutants displayed enhanced maximal effect, even though similar EC50 values suggested analogous agonist affinity (Table 1). Co-expression of the P2X3 mutants with Csk did not showed significant changes either in receptor tyrosine phosphorylation (n = 3, Fig. 3D) or current amplitude (Fig. 3E, n = 13). These data suggest that Tyr-393 was important to exploit the Csk modulation of P2X3 receptor functional response in an in vitro expression system. To understand the physiological implications of the interaction between Csk and P2X3 receptors, we studied mouse trigeminal sensory neurons that constitutively express P2X3 receptors potently modulated by NGF: indeed, NGF neutralization decreases trigeminal pain in vivo and P2X3 receptor currents in neuronal cultures (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar). It seemed, therefore, interesting to investigate the potential contribution by Csk signaling to the action of NGF. On trigeminal sensory neurons cultured in the presence of anti-NGF antibodies (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar), membrane expression of Csk was significantly enhanced by NGF neutralization (Fig. 4A) together with a stronger Csk activity as demonstrated by concomitant Src inhibition (see anti-phospho-Src527 antibody and Fig. 4A). To further validate Csk activation we examined, as a reliable index of this process, Csk translocation to the neuronal membrane (21Kawabuchi M. Satomi Y. Takao T. Shimonishi Y. Nada S. Nagai K. Tarakhovsky A. Okada M. Nature. 2000; 404: 999-1003Crossref PubMed Scopus (464) Google Scholar). Confocal microscopy showed, after NGF neutralization, redistribution of Csk to membrane (Fig. 4B). Quantification of microscopy experiments showed a significantly larger number of P2X3 receptor expressing neurons with a ring-like distribution of Csk (Fig. 4B, p = 0.009). Conversely, rapid disappearance of membrane-bound Csk was induced when exogenous NGF (100 ng/ml for 5 or 15 min) was applied to anti-NGF antibody-treated mouse trigeminal neurons (p = 0.02 n = 3, Fig. 4C), indicating a close relation between extracellular NGF levels and Csk location at the membrane level. Following neutralization of extracellular endogenous NGF with anti-NGF antibodies (24 h, 4), mouse trigeminal neurons showed a large increase in tyrosine phosphorylation of their P2X3 receptors (p = 0.03, Fig. 4D). The effects of NGF deprivation on P2X3 phosphorylation in culture were even more evident following tyrosine phosphatase inhibition with pervanadate (250 μm; 25 min; 0.79 ± 0.1 AU in control versus 2.24 ± 0.58 AU after pervanadate; n = 3; p < 0.001), suggesting that tyrosine phosphorylation rather than dephosphorylation was an important mechanism for the action by Csk on P2X3 receptors. On mouse trigeminal neurons, application of H89 (10 μm; 30 min) induced a slight increase of the constitutive N-terminal threonine phosphorylation of P2X3 receptors (0.24 ± 0.02 absolute gray levels for control and 0.26 ± 0.003 after H89 treatment, n = 3; supplemental Fig. S2B) previously described as attributed PKC activity (4D’Arco M. Giniatullin R. Simonetti M. Fabbro A. Nair A. Nistri A. Fabbretti E. J. Neurosci. 2007; 27: 8190-8201Crossref PubMed Scopus (80) Google Scholar). These results cannot exclude that, on trigeminal neurons, PKC or PKA should exert, at least partially, distinct and contrasting effects of the threonine and tyrosine phosphorylation state of P2X3 receptors. We tested the hypothesis that endogenous Csk exerts a constitutive control over P2X3 receptor function. Because transgenic mice lacking Csk are not vital (22Imamoto A. Soriano P. Cell. 1993; 73: 1117-1124Abstract Full Text PDF PubMed Scopus (347) Google Scholar), we examined our hypothesis by silencing Csk in HEK cells as well as in mouse trigeminal neurons (Fig. 5). Immunofluorescence and Western blotting demonstrated efficient silencing of Csk in HEK and in trigeminal ganglion cultures (n = 3, Fig. 5A and supplemental Fig. S2B). Western blotting with a phosphotyrosine antibody demonstrated that, in P2X3-expressing-HEK cells, there was no increase in P2X3 receptor tyrosine phosphorylation after silencing endogenous Csk (Fig. 5B), together with a significant rise in the maximal amplitude of the α,Β-meATP-evoked current with respect to responses measured from mock silenced cells (p = 0.04 for 10 μm and p = 0.02 for 100 μm α,Β-meATP, Fig. 5C). To explore any potential activity of residual Csk left after silencing in HEK cells, we decided to maximize the cAMP/PKA-mediated stimulation of endogenous residual Csk with forskolin application (10 μm, 5 min; known to be a strong Csk activator; Ref. 23Vang T. Torgersen K.M. Sundvold V. Saxena M. Levy F.O. Skålhegg B.S. Hansson V. Mustelin T. Taskén K. J. Exp. Med. 2001; 193: 497-507Crossref PubMed Scopus (272) Google Scholar) to either mock or siRNA-transfected HEK cells. Under these conditions, no significant change in P2X3 receptor phosphorylation or receptor current was found (supplemental Fig. S3), suggesting efficient silencing of endogenous Csk. We next tested the consequences of Csk silencing on the responses of P2X3 receptors of trigeminal neurons (Fig. 5D). Under these conditions, controlled experiments run in parallel with neurons treated with siRNA Csk or control non-targeting siRNA showed that the current amplitude evoked by 10 μm α,Β-meATP was significantly larger after Csk silencing (n = 29 in control and n = 24 cells for Csk siRNA; p = 0.04; Fig. 5D). These data indicate that endogenous Csk was essential to curtail constitutive, functional overactivation of P2X3 receptors in an expression system and in trigeminal neurons. The principal finding of the present study is the demonstration that the kinase Csk exerted a powerful inhibitory control on the function of P2X3 receptors via phosphorylation of the P2X3 Tyr-393 residue. These data shed light on a new mechanism that controls the efficiency of signaling by pain-sensing P2X3 receptors. It is tempting to sp