A Protein Kinase C Site Highly Conserved in P2X Subunits Controls the Desensitization Kinetics of P2X2 ATP-gated Channels

P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP receptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and voltage clamp recording inXenopus oocytes, we identified the highly conserved protein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (time constant, >1 min) rat P2X2receptors. Mutant receptors P2X2T18A, T18N, and K20T devoid of this consensus site exhibited quickly desensitizing properties (time constant, <1 s). In contrast with wild-type receptors, mutant P2X2 receptors with truncated C terminus exhibited variable cell-specific kinetics with quickly desensitizing currents converted to slowly desensitizing currents by phorbol ester-mediated stimulation of protein kinase C. Phosphorylation of Thr18 was demonstrated directly by immunodetection using specific monoclonal antibodies directed against the phosphothreonine-proline motif. Our data indicate that both phosphorylation of the conserved threonine residue in the N-terminal domain by protein kinase C and interaction between the two cytoplasmic domains of P2X2 subunits are necessary for the full expression of slowly desensitizing ATP-gated channels. P2X receptors are nonselective cation channels gated by extracellular ATP. Recombinant mammalian P2X subunits assemble in homomeric ionotropic ATP receptors that differ by their agonist sensitivity and desensitization rate in heterologous expression systems. Using site-directed mutagenesis and voltage clamp recording inXenopus oocytes, we identified the highly conserved protein kinase C site TX(K/R) located in the intracellular N terminus of P2X subunits as a critical determinant of kinetics in slowly desensitizing (time constant, >1 min) rat P2X2receptors. Mutant receptors P2X2T18A, T18N, and K20T devoid of this consensus site exhibited quickly desensitizing properties (time constant, <1 s). In contrast with wild-type receptors, mutant P2X2 receptors with truncated C terminus exhibited variable cell-specific kinetics with quickly desensitizing currents converted to slowly desensitizing currents by phorbol ester-mediated stimulation of protein kinase C. Phosphorylation of Thr18 was demonstrated directly by immunodetection using specific monoclonal antibodies directed against the phosphothreonine-proline motif. Our data indicate that both phosphorylation of the conserved threonine residue in the N-terminal domain by protein kinase C and interaction between the two cytoplasmic domains of P2X2 subunits are necessary for the full expression of slowly desensitizing ATP-gated channels. protein kinase A phorbol 12-myristate 13-acetate protein kinase C truncated Fast ionotropic responses to extracellular ATP are mediated by the activation of ATP-gated channels or P2X receptors present on the surface of various cell types. A family of genes coding for seven P2X channel subunits has been identified in human and rodents (1.North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (430) Google Scholar, 2.Buell G. Collo G. Rassendren F. Eur. J. Neurosci. 1996; 8: 2221-2228Crossref PubMed Scopus (241) Google Scholar). The electrophysiological characterization of recombinant homomeric and heteromeric P2X receptors expressed in heterologous systems led to their grouping in three functional categories based on their sensitivity to ATP and on their desensitization properties: 1) P2X1 (3.Valera S. Hussy N. Evans R.J. Adami N. North R.A. Surprenant A. Buell G. Nature. 1994; 371: 516-519Crossref PubMed Scopus (909) Google Scholar) and P2X3 (4.Chen C.-C. Akopian A.N. Sivilotti L. Colquhoun D. Burnstock G. Wood J.N. Nature. 1995; 377: 428-431Crossref PubMed Scopus (928) Google Scholar, 5.Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (897) Google Scholar) assemble in quickly desensitizing homomeric receptors highly sensitive to ATP and αβ-methylene ATP (EC50 around 1 μm); 2) P2X2 (6.Brake A.J. Wagenbach M.J. Julius D. Nature. 1994; 371: 519-523Crossref PubMed Scopus (847) Google Scholar), P2X4 (7.Buell G. Lewis C. Collo G. North R.A. Surprenant A. EMBO J. 1996; 15: 55-62Crossref PubMed Scopus (378) Google Scholar, 8.Séguéla P. Haghighi A. Soghomonian J.-J. Cooper E. J. Neurosci. 1996; 1: 448-455Crossref Google Scholar), P2X5 (9.Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar), P2X2+3 (5.Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (897) Google Scholar), P2X1+5 (10.Torres G.E. Haines W.R. Hegan T.M. Voigt M.M. Mol. Pharmacol. 1998; 54: 989-993Crossref PubMed Scopus (135) Google Scholar, 11.Lê K.-T. Boué-Grabot E. Archambault V. Séguéla P. J. Biol. Chem. 1999; 274: 15415-15419Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and P2X4+6 (12.Lê K.-T. Babinski K. Séguéla P. J. Neurosci. 1998; 18: 7152-7159Crossref PubMed Google Scholar) receptors are less sensitive to ATP (EC50 around 10 μm) and desensitize at slow to moderate rate; 3) P2X7 receptors (13.Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1542) Google Scholar) show low sensitivity to ATP (EC50 around 500 μm) and desensitize slowly. Modulation of the desensitization rate of neurotransmitter-gated channels is recognized as a potentially important mechanism for modulation of neuronal excitability (14.Smart T.G. Curr. Opin. Neurobiol. 1997; 7: 358-367Crossref PubMed Scopus (164) Google Scholar). P2X receptors are nonselective cation channels with high permeability to calcium ions (15.Rogers M. Dani J.A. Biophys. J. 1995; 68: 501-506Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 16.Ueno S. Koizumi S. Inoue K. Br. J. Pharmacol. 1998; 124: 1484-1490Crossref PubMed Scopus (19) Google Scholar), so the subtype-specific desensitization phenotype of ATP-mediated currents has a significant impact on the levels of intracellular calcium and subsequent activation of calcium-dependent effectors. Studies on the relationship between the slowly desensitization kinetics of P2X2receptor and its primary sequence have emphasized the requirement for several structural features including the transmembrane domains and their intracellular flanking regions (17.Werner P. Sewakdi E.P. Buell G.N. North R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15485-15490Crossref PubMed Scopus (130) Google Scholar), the negatively charged residue Asp349 located in the second transmembrane domain (18.Zhou Z. Monsma L.R. Hume R.I. Biochem. Biophys. Res. Commun. 1998; 252: 541-545Crossref PubMed Scopus (26) Google Scholar), and the unique C-terminal tail of the P2X2A splicing variant (19.Koshimizu T. Tomic M. Koshimizu M. Stojilkovic S.S. J. Biol. Chem. 1998; 273: 12853-12857Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20.Koshimizu T. Koshimizu M. Stojilkovic S.S. J. Biol. Chem. 1999; 274: 37651-37657Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 21.Smith F.M. Humphrey P.P.A. Murrell-Lagnado R.D. J. Physiol. 1999; 520: 91-99Crossref PubMed Scopus (40) Google Scholar). Chow and Wang (22.Chow Y.-W. Wang H.-L. J. Neurochem. 1998; 70: 2606-2612Crossref PubMed Scopus (42) Google Scholar) have reported that P2X2 receptor kinetics can also be modulated by protein kinase A (PKA)1-dependent phosphorylation of Ser431 located in the C-terminal domain of the subunit. However, no structural motif distinctive of the P2X family has been linked to a specific functional property of these ionotropic ATP receptors so far. Using site-directed mutagenesis and electrophysiological characterization of recombinant P2X receptors in Xenopusoocytes, we report here the identification of a phosphothreonine part of the highly conserved protein kinase C site located intracellularly in the N terminus of P2X subunits as a critical determinant for the expression of slowly desensitizing P2X2 ATP-gated channels. P2X2 receptor mutants were generated by polymerase chain reaction with the original P2X2 plasmid kindly provided by Dr D. Julius (University of California at San Francisco) (6.Brake A.J. Wagenbach M.J. Julius D. Nature. 1994; 371: 519-523Crossref PubMed Scopus (847) Google Scholar) as DNA template using PfuDNA polymerase (Stratagene) to minimize artifactual mutations. Point mutations were constructed using the QuickChange site-directed mutagenesis system (Stratagene) or the megaprimer mutagenic polymerase chain reaction. Briefly, primer containing one or several mismatch bases and a specific primer in the pcDNA3 vector (Invitrogen) sequence were used for the first amplification. The polymerase chain reaction product generated was used as megaprimer in a second round of amplification with an exact match oligonucleotide primers derived from P2X2 sequence. The mutated fragment thus obtained was cut with unique restriction enzymes and subcloned into the original P2X2 pcDNA3. For construction of C-terminal truncated receptors, we used a primer derived from amino acid sequence 368DKVRTPK374of P2X2 containing a single base mismatch to replace Thr372 by an alanine and followed by a terminalXhoI site. The full-length and truncated P2X2(wild-type and mutant forms) were then ligated with anXhoI-XbaI stuffer cassette containing in-frame His6 or FLAG epitope followed by an artificial stop codon in pcDNA3 vector to generate tagged P2X2 subunits as reported previously for other P2X subunits (11.Lê K.-T. Boué-Grabot E. Archambault V. Séguéla P. J. Biol. Chem. 1999; 274: 15415-15419Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 12.Lê K.-T. Babinski K. Séguéla P. J. Neurosci. 1998; 18: 7152-7159Crossref PubMed Google Scholar). All the constructs were subjected to automatic dideoxy sequencing (Sheldon Biotechnology Center, McGill University, Montreal, Canada). Ovary lobes were surgically retrieved from Xenopus laevis frogs under deep tricaine (Sigma) anesthesia. Oocyte-positive lobes were then treated for 3 h at room temperature with type I collagenase (Life Technologies, Inc.) in calcium-free Barth's solution under vigorous agitations. Stage V–VI oocytes were then manually defolliculated before nuclear microinjections of 1–5 ng of cDNA. The cells were maintained in Barth's solution containing 1.8 mm calcium chloride and 10 μg/ml gentamicin (Sigma) at 19 °C for up to 5 days. Two-electrode voltage clamp recordings were made 1–3 days after microinjection using an OC-725B amplifier (Warner Instruments). Signals were low pass filtered at 1 kHz, acquired at 500 Hz using a Macintosh IIci computer equipped with an NB-MIO-16XL analog-to-digital interface (National Instruments). Recorded traces were post-filtered at 20–50 Hz in Axograph (Axon Instruments). Ringer's solution containing 115 mm NaCl, 2.5 mm KCl, 1.8 mm CaCl2, and 10 mm HEPES buffered at pH 7.4 was perfused onto oocytes at a constant flow rate of 10–12 ml/min. Agonists and drugs were prepared in bath perfusion at their final concentration. ATP, PMA, 4αPMA, staurosporine, and chelerythine were purchased from Sigma. Drugs were initially dissolved in dimethyl sulfoxide before being diluted at least 1000-fold in bath solution to give the final working solution. Dose response curves were fitted to the Hill sigmoidal equation and EC50 values were determined by nonlinear regression analysis using the Prism 2.0 software (Graphpad, San Diego, CA). Desensitization rates were measured during 10-s applications of 100 μm ATP. For the desensitization of truncated mutants, both the peak current and the current amplitude 5 s after the peak (sustained current phase) were measured, and desensitization is expressed as the peak/sustained current ratio. We used unpairedt test to compare desensitization rates, and statistical significance was set at p < 0.05. cDNA transfections of FLAG-tagged P2X2 subunits were performed in mammalian HEK cells using the calcium phosphate method as described previously (11.Lê K.-T. Boué-Grabot E. Archambault V. Séguéla P. J. Biol. Chem. 1999; 274: 15415-15419Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 12.Lê K.-T. Babinski K. Séguéla P. J. Neurosci. 1998; 18: 7152-7159Crossref PubMed Google Scholar). Transfected HEK-293A cells or injected oocytes used for Western blots were collected in phosphate-buffered saline pelleted at low centrifugation and homogenized in 10 volumes of 10 mm HEPES buffer and 0.3 m sucrose, pH 7.4, containing a protease inhibitor mixture (Sigma) and phosphatase inhibitors (1 μm microcystin-LR (Calbiochem), 25 mm sodium fluoride, 5 mm sodium pyrophosphate). Membranes from cell lysates were solubilized with 1% Triton X-100 for 2 h at 4 °C and pelleted at 14,000 × g for 5 min. Membrane proteins within supernatants were used for Western blots. Solubilized proteins were incubated with 100 μl of equilibrated anti-FLAG M2 affinity gel (Sigma) for 2 h at 4 °C under agitation. Then resin beads were washed three times in Tris-buffered saline containing 1% Triton X-100. Bound proteins were eluted from the anti-FLAG M2 affinity gel with 0.1 m glycine, pH 3.5. Samples were then loaded onto 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Immunostainings were performed first with an anti-phosphothreonine-proline monoclonal antibody (1:500) (New England Biolabs) followed by incubation with anti-mouse peroxidase-labeled secondary antibodies (1:2,000) for visualization by enhanced chemiluminescence (Amersham Pharmacia Biotech). Membranes were then stripped by 30 min of incubation at 50 °C in a solution of 150 mm Tris, pH 7.5, 2% (w/v) SDS, 0.8% (v/v) β-mercaptoethanol and washed four times in Tris-buffered saline before staining with anti-FLAG M2 murine monoclonal antibody (1:1000) (Sigma) followed by incubation with anti-mouse peroxidase-labeled secondary antibodies (1:5,000) for visualization by enhanced chemiluminescence. Members of an unique class of ligand-gated channels by their protein topology, P2X ATP receptors are produced by the oligomeric assembly of two-transmembrane domain subunits with both their N and C termini located intracellularly (23.Torres G.E. Hegan T.M. Voigt M.M. FEBS Lett. 1998; 425: 19-23Crossref PubMed Scopus (58) Google Scholar). All known P2X ATP-gated channel subunits display a threonine residue that is part of the highly conserved protein kinase C consensus site TX(K/R) in the intracellular N-terminal domain of the protein (Fig.1 A). To test the role of this conserved domain in the function of P2X2 ATP-gated channels, we first substituted Thr18 for Ala in the sequence of rat P2X2 subunit to eliminate this protein kinase C site. Following expression of this mutant P2X2T18A receptor in Xenopus oocytes, we observed a dramatic change of phenotype in response to application of extracellular ATP. In contrast with wild-type P2X2 receptors that do not desensitize significantly over the time of application of agonist, mutant P2X2T18A subunits assembled in functional ATP-gated channels that displayed fast kinetics with complete desensitization in less than 5 s (Fig. 1 B). Despite a short time of recovery (Fig.2 A), the rate of desensitization of P2X2T18A was faster than the rate of P2X1 receptors expressed in oocytes (Fig. 2 B). We measured a time constant (50% of maximal response) of 0.36 s ± 0.11 for P2X2T18A and 0.80 s ± 0.11 for P2X1 receptors. of desensitization rate be linked to structural that the agonist sensitivity of the mutant channels. of the agonist an in the of the subunits also an change of voltage sensitivity to kinetics at specific we the of the agonist and of the by recording the of P2X2T18A channels at of ATP 1 μm to 1 and at to significant in the desensitization rate (Fig. 2 A). that an of sensitivity to agonist was not the mechanism the the EC50 of ATP for P2X2T18A was to be ± 2 with an EC50 of ± wild-type P2X2 receptors (Fig. 2 For the EC50 measured for P2X2T18A be of the fast kinetics of desensitization of these mutant receptors. However, in the of agonist (1 μm to 1 we not a change of phenotype at high of ATP in the of wild-type seven to times sensitive than P2X2T18A mutant receptors. We to eliminate the protein kinase C site by the conserved Thr18 by of Ala to the observed on the kinetics be to a structural change to the of a phosphorylation site. The mutant receptor the quickly desensitizing kinetics than P2X2T18A also that this threonine residue an unique structural role of being a phosphate so we the protein kinase C site by the charged that is a second critical part of the kinase site by the conserved The mutant receptor displayed the fast phenotype than P2X2T18A and receptors (Fig. To test the of the structural of the protein kinase C motif in P2X2 we receptors generated slowly desensitizing currents in response to ATP, to observed with wild-type channels (Fig. The of these that the of the protein kinase C motif is a critical determinant of the kinetics of wild-type the that also a structural rat P2X2 subunits have a intracellular C-terminal domain that two other protein kinase C on Thr372 and and one protein kinase A site on Ser431 (22.Chow Y.-W. Wang H.-L. J. Neurochem. 1998; 70: 2606-2612Crossref PubMed Scopus (42) Google Scholar). splicing of P2X2 subunits display of these protein kinase and several have that the of the C-terminal domain in these a role on the desensitization kinetics of P2X2 channels (19.Koshimizu T. Tomic M. Koshimizu M. Stojilkovic S.S. J. Biol. Chem. 1998; 273: 12853-12857Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20.Koshimizu T. Koshimizu M. Stojilkovic S.S. J. Biol. Chem. 1999; 274: 37651-37657Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 21.Smith F.M. Humphrey P.P.A. Murrell-Lagnado R.D. J. Physiol. 1999; 520: 91-99Crossref PubMed Scopus (40) Google Scholar). To eliminate these phosphorylation we produced truncated of wild-type and mutant P2X2 subunits the C-terminal sequence from to the codon and mutated also on Thr372 to eliminate the protein kinase C site. of the C-terminal domain in receptors not the critical of intracellular domains in the function of ATP-gated channels. in truncated P2X2 receptors with N-terminal domains displayed two a phenotype of the oocytes, three with a quickly desensitizing followed by a slowly desensitizing or a slowly desensitizing phenotype (Fig. the truncated P2X2 receptors the conserved site displayed a quickly desensitizing phenotype to the one observed with full-length P2X2T18A mutant receptors 1 and of the truncated the of cell-specific intracellular that be with of in the levels of protein kinase in oocytes K. P. J. Neurosci. 1998; 18: PubMed Google Scholar). The functional of the C-terminal domain is also demonstrated by the of current between the full-length and the truncated of P2X2T18A and mutant channels. The of peak currents than 500 at 100 μm obtained with truncated P2X2T18A or K20T mutants (Fig. that a specific intracellular interaction the assembly and surface expression of ATP-gated channels. the variable phenotype observed with truncated is linked to variable in oocytes by the with phorbol we be to a on the mutant a of the desensitization rate and less in the kinetics of To test this we the oocytes wild-type truncated or with the phorbol or with its Thr18 is a phosphate in a protein kinase C currents generated by truncated P2X2 channels were converted into slowly desensitizing currents of the peak amplitude in of not in a of the phorbol desensitizing wild-type P2X2 receptors or mutant devoid of N-terminal site were to phorbol (Fig. and to inhibitors of μm and data not the low of wild-type P2X2 phenotype and the of sensitivity to stimulation or be with the phosphorylation of Thr18 by in oocytes as as in mammalian cells F.M. Humphrey P.P.A. Murrell-Lagnado R.D. J. Physiol. 1999; 520: 91-99Crossref PubMed Scopus (40) Google Scholar, Y.-W. Wang H.-L. J. Neurochem. 1998; 70: 2606-2612Crossref PubMed Scopus (42) Google Scholar). The that wild-type P2X2 receptors do not display a in their phenotype in heterologous systems that the C-terminal domain an important role in the of the slowly desensitization In C terminus domain phosphorylation of the subunit subunits from phosphatase in of the C terminus the expression of a slowly desensitizing phenotype the stimulation of to the P2X2 To directly the phosphorylation of Thr18 in Western from oocytes and cells P2X2 receptors, we used specific monoclonal antibodies directed against the phosphothreonine-proline motif that be present the conserved site of the receptor subunit is affinity of FLAG P2X2 receptors, we a to wild-type P2X2 receptors, no was observed in the of mutant receptors the N-terminal site in oocytes not as as in cells A). We for levels of receptor expression in the with anti-FLAG epitope antibodies (Fig. B). We the mutant as in this in recordings we currents to its high of expression (Fig. The of a single phosphothreonine that the conserved site present in the N-terminal domain of P2X2 channel subunit is and that the subunits are on Thr18 in oocytes and cells heterologous systems. The of in the mutant also that the present in the C-terminal domain of the subunit is not We from these data that in the N-terminal site of the P2X2 subunit is a critical determinant for the slow rate of desensitization of homomeric P2X2 ATP-gated channels intracellular to be in oocytes as as in cells heterologous expression systems. The of in mutants devoid of the conserved site ATP receptors with fast kinetics of that the expression of receptors with a slow rate of desensitization specific between the intracellular domain and other channel Despite its a full-length C-terminal domain is not necessary for the expression of P2X2 slowly desensitizing domain to a role on the slow phenotype an intracellular and to have on the response of P2X2 receptors to Ser431 phosphorylation by P2X2 currents by the rate of desensitization of the channels (22.Chow Y.-W. Wang H.-L. J. Neurochem. 1998; 70: 2606-2612Crossref PubMed Scopus (42) Google Scholar), by a response by their rate of phosphorylation has previously been reported to neuronal excitability the modulation of channels for by chloride channels S. P. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.G. 1994; Full Text PDF PubMed Scopus Google Scholar) and directly by receptor currents L. Nature. PubMed Scopus Google Scholar). In a of primary P2X2 subunits with P2X3 subunits to a heteromeric ATP-gated channel with the slowly desensitization kinetics of homomeric P2X2 receptors and the high sensitivity to ATP and αβ-methylene ATP of homomeric P2X3 receptors (5.Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-435Crossref PubMed Scopus (897) Google Scholar, J. Neurosci. PubMed Google Scholar). of ATP on in G. 1996; PubMed Scopus Google Scholar), the exact role of P2X receptors in is not The of an and critical site on P2X2 subunits that the desensitization kinetics of P2X currents be to second the levels of and and or receptors to C activation the to extracellular ATP the of the desensitization rate of P2X channels. The N-terminal site TX(K/R) is conserved in known P2X P2X subunits assembled in ATP receptors with slowly desensitizing or quickly desensitizing are and to heterologous functional to be We and for their as as on P2X2T18A

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