Differential Assembly of Rat Purinergic P2X7 Receptor in Immune Cells of the Brain and Periphery

ATP-gated P2X7 purinoceptors are found in most immune cells of the periphery and the brain where their activation leads to multiple downstream events such as cell permeabilization, apoptosis, and/or cytokine release. P2X7receptors do not form heteromeric receptors with any of the other six P2X subunits, and it is not known what type of homomeric assemblies the P2X7 subunit makes. We constructed and purified an ectodomain protein of the rat P2X7 receptor (amino acids 60–323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X7 receptors in central and peripheral immune cells. In HEK cells expressing rat P2X7receptors, the Ab increased the maximum current evoked by BzATP by 3–8-fold with a 5-fold leftward shift in EC50concentration. This Ab recognized only a non-denatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X7 Ab recognized both monomeric subunits on SDS-PAGE and a multimeric complex on blue native-PAGE in this heterologous expression system. With Western blotting using these two Abs, native P2X7 receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X7 receptors in brain glia and/or astrocytes appear to form only as monomeric subunits. ATP-gated P2X7 purinoceptors are found in most immune cells of the periphery and the brain where their activation leads to multiple downstream events such as cell permeabilization, apoptosis, and/or cytokine release. P2X7receptors do not form heteromeric receptors with any of the other six P2X subunits, and it is not known what type of homomeric assemblies the P2X7 subunit makes. We constructed and purified an ectodomain protein of the rat P2X7 receptor (amino acids 60–323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X7 receptors in central and peripheral immune cells. In HEK cells expressing rat P2X7receptors, the Ab increased the maximum current evoked by BzATP by 3–8-fold with a 5-fold leftward shift in EC50concentration. This Ab recognized only a non-denatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X7 Ab recognized both monomeric subunits on SDS-PAGE and a multimeric complex on blue native-PAGE in this heterologous expression system. With Western blotting using these two Abs, native P2X7 receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X7 receptors in brain glia and/or astrocytes appear to form only as monomeric subunits. human embryonic kidney benzoyl-benzoyl-ATP antibody enzyme-linked immunosorbent assay phosphate-buffered saline polyacrylamide gel electrophoresis blue native-PAGE polyvinylidene difluoride thioredoxin Like most neurotransmitters, extracellular ATP activates both metabotropic (G protein-coupled) receptors, the P2Y receptor family, and ionotropic (ligand-gated) receptors, the P2X receptor family (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (430) Google Scholar). When compared with other ionotropic receptors (e.g.nicotinic), which are primarily or solely expressed in nerve and muscle, P2X receptors are unusual in their wide spread expression in both excitable and non-excitable cells (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (430) Google Scholar, 2MacKenzie A.B. Surprenant A. North R.A. Ann. N. Y. Acad. Sci. 1998; 868: 716-729Crossref Scopus (89) Google Scholar, 3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). There are seven P2X receptors, six of which (P2X1–6) are found in brain and peripheral neurons; additionally, mRNA for P2X1, P2X4, and the non-neuronal P2X7 receptor are prominent in many immune cells, particularly monocytes, macrophage, bone marrow, and brain microglia (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (430) Google Scholar, 2MacKenzie A.B. Surprenant A. North R.A. Ann. N. Y. Acad. Sci. 1998; 868: 716-729Crossref Scopus (89) Google Scholar, 3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). The P2X7 receptor shows 40–45% amino acid identity with any one of the other P2X receptors; it shares the overall membrane topology of two transmembrane domains, intracellular N and C termini, and the large ectodomain with conservation of the 10 extracellular cysteine residues, although its C-terminal is some 200 amino acids longer than other P2X receptors (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (430) Google Scholar, 2MacKenzie A.B. Surprenant A. North R.A. Ann. N. Y. Acad. Sci. 1998; 868: 716-729Crossref Scopus (89) Google Scholar, 3Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar). However, the functional sequelae of P2X7 receptor activation differ strikingly from other P2X receptors. Activation of P2X7 receptors expressed in mammalian cells, such as HEK1 or CHO cells, opens both a small cationic channel and a large (up to 900 Da) dye-permeable pore, followed within seconds by extensive membrane blebbing (4Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1545) Google Scholar, 5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (329) Google Scholar); similar events occur upon activation of native P2X7 receptors in some but not all immune cells expressing this receptor (6Di Virgilio F. Sanz J.M. Chiozzi P. Falzoni S. Prog. Brain Res. 1999; 120: 355-368Crossref PubMed Google Scholar, 7Di Virgilio F. Chiozzi P. Falzoni S. Ferrari D. Sanz J.M. Venketaraman V. Baricordi O.R. Cell Death Differ. 1998; 5: 191-199Crossref PubMed Scopus (229) Google Scholar, 8Markwardt F. Lohn M. Bohm T. Klapperstuck M. J. Physiol. 1997; 498: 143-151Crossref PubMed Scopus (53) Google Scholar). The mechanism(s) underlying formation of the permeabilizing pore and other downstream events such as membrane blebbing are not known but the intracellular C-terminal domain is required (4Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1545) Google Scholar). Moreover, whereas each of the other P2X receptors is capable of forming heteromeric proteins with various other subunits in a specific pattern, the P2X7subunit cannot heteropolymerize with any of the other subunits (9Torres G.E. Egan T.M. Voigt M.M. J. Biol. Chem. 1999; 274: 6653-6659Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), even with the P2X4 receptor with which it co-exists in monocytes, bone marrow, and many endothelia and epithelia (4Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1545) Google Scholar, 10Buell G. Lewis C. Collo G. North R.A. Surprenant A. EMBO J. 1996; 15: 55-62Crossref PubMed Scopus (378) Google Scholar). Biochemical evidence to date favors a trimeric stoichiometry for P2X receptors expressed in oocytes although higher multiples of a trimeric architecture cannot be ruled out (11Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (490) Google Scholar, 12Buttner, C., Sadtler, A., Aschrafi, A., Markwardt, F., and Schmalzing, G. (2000) Purines 2000 International Symposium on Nucleosides and Nucleotides, Madrid (Abstr. 40).Google Scholar). Other biochemical, biophysical, and mutagenesis data based on experiments with homomeric P2X2 receptors are compatible with either a trimeric or tetrameric stoichiometry (13Ding S. Sachs F. J. Gen. Physiol. 1999; 113: 695-720Crossref PubMed Scopus (148) Google Scholar, 14Stoop R. Thomas S. Rassendren F. Kawashima E. Buell G. Surprenant A. North R.A. Mol. Pharmacol. 1999; 56: 973-981Crossref PubMed Scopus (117) Google Scholar, 15Kim M. Yoo O.J. Choe S. Biochem. Biophys. Res. Commun. 1997; 240: 618-622Crossref PubMed Scopus (62) Google Scholar). No information yet exists on the multimeric nature of native P2X7 receptors. We have expressed and purified a protein comprising the extracellular domain of the rat P2X7 receptor and have used this protein to generate an ectodomain-specific monoclonal Ab to better probe membrane localization of native P2X7 receptors in both peripheral and brain immune cells. Our studies have revealed striking differences in P2X7 receptor assembly in cells of the peripheral immune system and the brain immune system. A bacterial expression vector, designated pP2X7ecto, was constructed by ligation of a 792-base pair fragment from a polymerase chain reaction product, comprising amino acids 60–323 of rat P2X7 (GenBankTM NM019256) into vector pET32 (Novagen). The plasmid construct was verified by DNA sequencing with BigDye terminator cycle sequencing kit (PE Applied Biosystems). BL21(DE3) Escherichia coli transformed with pP2X7ecto was used for overexpression of ecto-rP2X7 protein. After overnight induction of protein expression by 1 mm IPTG (isopropyl-β-d-thiogalactopyranoside), the culture was centrifuged at 5,000 × g for 10 min. The bacterial pellet was resuspended (1:50 of the culture volume) in lysis buffer (50 mm Tris, pH 8.0, 200 mm NaCl, 2% Triton X-100) and subjected to lysis by sonication on ice. The protein, aggregated in inclusion bodies, was pelleted by centrifugation at 10,000 ×g for 20 min. The pellet was extracted once with 1:5 of the resuspension buffer (200 mm NaCl, 50 mm Tris, 2m urea, Triton X-100, at pH 8.0) by vigorous vortexing for 10 min. The extracted pellet was solubilized in 1:20 of 8 murea buffer (8 m urea, 50 mm Tris, pH 8.0, 200 mm NaCl, 1% SDS, 10% glycerol), centrifuged at 15,000 × g for 30 min. The solubilized protein was purified and refolded by Ni2+-affinity chromatography followed by thrombin digestion and gel filtration chromatography by Superdex 200 (Amersham Pharmacia Biotech). Protein concentration was determined by protein assay kits (Bio-Rad) with bovine serum albumin used as the standard. Monoclonal antibodies were generated against ecto-rP2X7 protein by Sheffield Hybridomas (University of Sheffield). Primary screening was performed by immunohistochemistry on stably transfected HEK cells and by ELISA. Ecto-P2X7Ab was purified from hybridoma supernatant on a protein G-Sepharose affinity chromatography column. HEK cells stably transfected with rP2X7 or rP2X2 (5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (329) Google Scholar) were used for these experiments. To prepare whole-cell lysates, the cells were freeze-thawed three times and sonicated. For isolation of membrane fractions, the cells were grown in T75 flasks, washed with ice-cold PBS, and harvested. After brief centrifugation, the cells were resuspended in hypotonic lysis buffer containing 10 mmTris-HCl (pH 7.5) and protease inhibitors (CompleteTM,Roche Molecular Biochemicals). After incubation on ice for 10 min, the cell suspension was homogenized by a Dounce homogenizer, adjusted to 155 mm NaCl, and subjected to ultracentrifugation at 60,000 × g for 1 h at 4 °C. The pellet (membrane fraction) was solubilized in solubilization buffer (20 mm dodecylmaltoside in PBS containing protease inhibitor) for 3 h at 4 °C. Adult Wistar rats (43–45 days old) were used in all experiments. They were killed by excess CO2, brains rapidly removed, and placed in ice-cold PBS containing protease inhibitor. The cerebellum and hippocampi were dissected out and 400-μm thick coronal were using a The of each were in PBS, of and and to for into a containing solubilization were from peritoneal of were in containing 10% serum and washed in were by marrow cells were from and washed three times with ice-cold bone marrow cells and peritoneal were with 1 for 1 h and the marrow cells, peritoneal and brain were extracted with solubilization buffer overnight at 4 °C. The were centrifuged for 30 at 4 °C. The supernatant was to SDS-PAGE or with was used at a concentration of proteins were by SDS-PAGE and to polyvinylidene difluoride were in buffer (20 mm Tris, mm NaCl, pH 7.5) and with buffer containing for 1 After with (20 mm Tris, mm NaCl, pH the were overnight with polyclonal antibody polyclonal antibody or ecto-rP2X7 Ab in antibody buffer (20 mm Tris, mm NaCl, pH After multiple with the were for 1 h with or antibody were and proteins were with assay kit (Bio-Rad) the was performed as (11Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (490) Google Scholar, G. Biochem. PubMed Scopus Google Scholar) with the membrane were with buffer to a concentration of 10% and 50 mm adjusted with to pH For the of was used in 50 mm pH Molecular Molecular were to two on both of the polyacrylamide from 4 to or gel were performed at 4 and were to and with experiments were times with similar were with and with in m were removed, for a 3 and placed in in PBS to the brains to The brains were at a of and as in m saline pH were in containing and for with of with a containing either serum for polyclonal or serum for monoclonal in with Triton for 1 were for 4 days at 4 in the containing Ab was performed either in the of at the concentration as or in the of in with polyclonal antibodies were in and with monoclonal antibodies were in at a of of with either 2% or Triton X-100, bovine serum albumin for 4 h at was by of the in for min. A similar was used for peritoneal macrophage or bone marrow cells but incubation in antibodies (20 was For HEK cells, was in serum in PBS with 2% Triton and with Ab for h at or overnight at 4 °C. were and the Ab for 1 cells were with in serum in PBS and with Ab for h at with Ab for 1 The were polyclonal all from monoclonal from and rat Ab cell were out as (4Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1545) Google Scholar, 5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (329) Google Scholar). were in stably transfected cells expressing rat P2X7 by of BzATP for at were and or incubation with or 4 Ab at °C. 1 shows the in the purified ectodomain protein. The thioredoxin protein be as a at the of The affinity by affinity in a this digestion in the two protein and in the was purified by gel filtration chromatography to and of This a protein on the SDS-PAGE gel as the at the of This protein was used to generate the ecto-rP2X7 monoclonal Ab used in the When the Ab was was in HEK cells with in HEK cells expressing any other rat or human P2X in the human We were to in HEK cells expressing the human P2X7 receptor with the polyclonal C-terminal P2X7 receptor Ab although functional expression of this receptor was by not The of the rat P2X7 receptor was in and cells, whereas the C-terminal P2X7 receptor Ab only cells To the Ab functional of this membrane in to the ATP from cells with a Ab with the C-terminal P2X7 Ab or with the from cells with either the serum or the polyclonal P2X7 Ab were not from from HEK cells 4 for However, incubation with Ab for 30 or increased the maximum current evoked by BzATP and the by to the incubation times (up to 3 of whereas the not of the to its these a functional Ab receptor in the form of an of the membrane current and an in in affinity at the have of for in extracellular or or to ATP all shift the to the Pharmacol. 1999; PubMed Scopus Google Scholar, Buell G. J. Physiol. PubMed Google Scholar). We any into the multimeric nature of the expressed P2X7 receptor by the of the Ab and the C-terminal P2X7 Ab in Western using both denatured and on and We used HEK cells expressing rat P2X2 receptors and a C-terminal P2X2 polyclonal Ab as a The three antibodies were all specific for their proteins The P2X2 which used as a for two from HEK cells, at and these are the as for and P2X2 receptors expressed in either HEK cells or oocytes G.E. Egan Voigt M.M. 1998; PubMed Scopus Google Scholar, A. R. C. Buell G. Surprenant A. North R.A. Rassendren F. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the C-terminal P2X7 Ab recognized the monomeric P2X7 on it an multimeric form A and However, the Ab did not recognize the denatured P2X7 receptor on but strongly bound to the form on and The a at which was not by the C-terminal polyclonal P2X7 Ab 3 and In with with the C-terminal polyclonal P2X7 Ab the Ab the human P2X7 receptor on SDS-PAGE or on not We performed similar and Western blotting experiments on rat bone marrow, peritoneal macrophage, and We strongly denatured or denatured to similar or were was in peritoneal macrophage using either the Ab or the C-terminal polyclonal 4 The C-terminal P2X7 Ab in non-neuronal cells in the as as the cerebellum 4 However, of any brain was with the Ab although and were The C-terminal P2X7 Ab two at and from and these were from either by or The is with the form of the P2X7 receptor C., Sadtler, A., Aschrafi, A., Markwardt, F., and Schmalzing, G. (2000) Purines 2000 International Symposium on Nucleosides and Nucleotides, Madrid (Abstr. 40).Google Scholar) and was in HEK cells expressing rat P2X7 receptors. When peritoneal macrophage or bone marrow were strongly the C-terminal P2X7 Ab the with evidence of the a similar was for peritoneal macrophage However, it an from bone marrow cells with only The Ab did not any of the strongly denatured on SDS-PAGE to the C-terminal it the from bone marrow on SDS-PAGE only was used but from peritoneal macrophage or brain on the C-terminal Ab an from both peritoneal macrophage and bone marrow but only a from and The Ab a from both peritoneal macrophage and bone marrow, but from brain The from this is native P2X7 receptors appear to exist as a strongly bound multimeric complex in peripheral bone marrow and peritoneal macrophage but only as monomeric in brain cells. Our on from of denatured and the of a C-terminal polyclonal Ab and a monoclonal ectodomain Ab on P2X7 receptors expressed in HEK cells and on native P2X7 receptors in the brain and of brain and peritoneal macrophage for We in some the of the used in by and by Western blotting on HEK cell expressing the of other P2X receptors as as HEK cells and be these are both and specific for rat expressed P2X7receptors The Ab bound the functional receptor in HEK cells, to the current and shift the BzATP concentration to the was the in both and cells using the whereas the polyclonal C-terminal P2X7 Ab only cells. are in with on other P2X receptors, primarily the P2X2 which have the C to the intracellular of the membrane (9Torres G.E. Egan T.M. Voigt M.M. J. Biol. Chem. 1999; 274: 6653-6659Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 14Stoop R. Thomas S. Rassendren F. Kawashima E. Buell G. Surprenant A. North R.A. Mol. Pharmacol. 1999; 56: 973-981Crossref PubMed Scopus (117) Google Scholar, G.E. Egan T.M. Voigt M.M. 1998; PubMed Scopus Google Scholar), and the Ab to the extracellular domain of the functional receptor in these cells. of membrane proteins information subunit and the nature of the multimeric by and receptors (11Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (490) Google Scholar, G. Biochem. PubMed Scopus Google Scholar, N. C. A. J. Schmalzing G. EMBO J. 1999; PubMed Scopus Google Scholar). In Schmalzing and (11Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (490) Google C., Sadtler, A., Aschrafi, A., Markwardt, F., and Schmalzing, G. (2000) Purines 2000 International Symposium on Nucleosides and Nucleotides, Madrid (Abstr. 40).Google Scholar) have this to homomeric P2X1, and P2X7 receptors expressed in where with denatured and P2X receptor proteins a trimeric We have not the of stoichiometry in but have used to native P2X7 receptors form as multimeric do in peritoneal macrophage and bone marrow, as revealed by both C-terminal and Abs, but do not in brain cells 3 The on the is not with a receptor of 3 or 4 subunits and a receptor of or subunits. However, the this complex not only the P2X7 receptor but one or The of this Ab of such proteins affinity and PubMed Scopus Google Scholar). In any the from these experiments is brain P2X7receptors exist as and do not form a multimeric assembly are from brain evidence for this from where the C-terminal P2X7 Ab only a monomeric from and evidence this from the of any of these brain on either or SDS-PAGE by the Ab and the of of brain by the Ab whereas the C-terminal Ab cells in brain by immunohistochemistry and recognized monomeric receptor on SDS-PAGE as on are with from rat P2X7 receptors expressed in HEK cells, where the C-terminal P2X7 Ab recognized both monomeric and multimeric of the receptor whereas the Ab recognized only the multimeric but not the monomeric form of the receptor We differences in the two antibodies or the other differences in P2X7 receptor assembly or and/or antibody a at in HEK cells on with the a was not in native the from a complex, it is the a trimeric receptor as to be the for P2X7 receptors expressed in oocytes where the on is at C., Sadtler, A., Aschrafi, A., Markwardt, F., and Schmalzing, G. (2000) Purines 2000 International Symposium on Nucleosides and Nucleotides, Madrid (Abstr. 40).Google Scholar). with the to both recognized a from bone marrow on SDS-PAGE whereas the from the other peritoneal macrophage, did not this be a form of the P2X7 receptor from other proteins or P2X7 subunits are by cannot for these with the evidence for multimeric assembly in peripheral immune cells in to monomeric assembly in brain it is to assembly of P2X7 receptor be the than the studies of P2X7 receptors from human Buell G. J. Physiol. PubMed Google Scholar, R.A. R. P. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and peripheral Buell G. J. Physiol. PubMed Google Scholar) have shown or as a of a a of the protein is However, the and/or functional be increased by in extracellular or by into macrophage Buell G. J. Physiol. PubMed Google Scholar). the monomeric form of the P2X7 receptor from brain be to a intracellular this be with on Western of cerebellum and where two and were The the form of the a an intracellular and not or protein. The of the assembly of P2X7 receptors brain and peripheral immune cells in the to be but it is of the differences in the functional sequelae of P2X7 receptor activation have for various For activation of P2X7 receptors expressed in HEK cells in formation of a pore to of to 900 (4Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1545) Google Scholar, 5Virginio C. MacKenzie A.B. North R.A. Surprenant A. J. Physiol. 1999; 519: 335-346Crossref PubMed Scopus (329) Google Scholar, Virgilio F. Sanz J.M. Chiozzi P. Falzoni S. Prog. Brain Res. 1999; 120: 355-368Crossref PubMed Google Scholar) although expression in oocytes not S. M. 1997; PubMed Scopus Google Scholar). P2X7 receptors in and cells to a dye-permeable pore formation (6Di Virgilio F. Sanz J.M. Chiozzi P. Falzoni S. Prog. Brain Res. 1999; 120: 355-368Crossref PubMed Google Scholar, 7Di Virgilio F. Chiozzi P. Falzoni S. Ferrari D. Sanz J.M. Venketaraman V. Baricordi O.R. Cell Death Differ. 1998; 5: 191-199Crossref PubMed Scopus (229) Google Scholar) but P2X7 receptors in or not on the or activation of the cells F. Lohn M. Bohm T. Klapperstuck M. J. Physiol. 1997; 498: 143-151Crossref PubMed Scopus (53) Google Scholar, Buell G. J. Physiol. PubMed Google Scholar, M. C. Bohm T. Schmalzing G. F. Biophys. PubMed Scopus Google Scholar, F. Klapperstuck M. Lohn M. D. C. Schmalzing G. Prog. Brain Res. 1999; 120: PubMed Google Scholar). cells or cell form these large upon P2X7 activation D. Chiozzi P. Falzoni S. M. Collo G. Buell G. Virgilio F. 1997; PubMed Scopus Google Scholar, D. Chiozzi P. Falzoni S. S. Virgilio F. J. 1997; PubMed Scopus Google Scholar), but it is not known this in in brain Our in the was to P2X7 receptors from peripheral and central immune cells. the striking differences have it be to the functional of P2X7 receptors in microglia in brain C. C. J. PubMed Scopus Google Scholar, 1999; Google Scholar), which was the of brain to the and of P2X7receptors in the cells 1999; Google Scholar, T. J. F. P. C. P. Burnstock G. A. J. PubMed Google Scholar, T. Y. S. P. S. J. Pharmacol. 1998; PubMed Scopus Google Scholar). be to the monomeric assembly in brain immune cells and/or the multimeric assembly in peripheral immune cells or We and for cell