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An Ile-568 to Asn Polymorphism Prevents Normal Trafficking and Function of the Human P2X7 Receptor

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m212759200

1083-351X

James S. Wiley, Lan‐Phuong Dao‐Ung, Changping Li, Anne N. Shemon, Ben J. Gu, Megan L. Smart, Stephen J. Fuller, Julian A. Barden, Steven Petrou, Ronald Sluyter,

Bipolar Disorder and Treatment

The P2X7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X7 receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X7 function was measured by ATP-induced fluxes of Rb+, Ba2+, and ethidium+ into various lymphocyte subsets and was decreased to values of ∼257 of normal. The expression of the P2X7 receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X7 carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-γ up-regulated P2X7 function in cells heterozygous for the Ile-568 to Asn mutation to a value around 507 of normal. These data identify a second loss-of-function polymorphism within the P2X7 receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581. The P2X7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X7 receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X7 function was measured by ATP-induced fluxes of Rb+, Ba2+, and ethidium+ into various lymphocyte subsets and was decreased to values of ∼257 of normal. The expression of the P2X7 receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X7 carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-γ up-regulated P2X7 function in cells heterozygous for the Ile-568 to Asn mutation to a value around 507 of normal. These data identify a second loss-of-function polymorphism within the P2X7 receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581. lipopolysaccharide chronic lymphocytic leukemia fluorescein isothiocyanate monoclonal antibody(ies) human embryonic kidney natural killer The purinergic P2X7 receptor is a ligand-gated channel, selective for cationic permeants, which has a wide distribution including cells of the immune and hemopoietic system (1Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Crossref PubMed Scopus (616) Google Scholar,2North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2438) Google Scholar). Activation of this receptor by brief exposure to extracellular ATP opens a channel that allows Ca2+ and Na+ influx and K+ efflux and that initiates a cascade of intracellular downstream events. These include the stimulation of phospholipase D (3El-Moatassim C. Dubyak G.R. J. Biol. Chem. 1993; 268: 15571-15578Abstract Full Text PDF PubMed Google Scholar,4Gargett C.E. Cornish E.J. Wiley J.S. Biochem. J. 1996; 313: 529-535Crossref PubMed Scopus (65) Google Scholar), the activation of membrane metalloproteases (5Jamieson G.P. Snook M.B. Thurlow P.J. Wiley J.S. J. Cell. Physiol. 1996; 166: 637-642Crossref PubMed Scopus (84) Google Scholar, 6Gu B. Bendall L.J. Wiley J.S. Blood. 1998; 92: 946-951Crossref PubMed Google Scholar, 7Sluyter R. Wiley J.S. Int. Immunol. 2002; 14: 1415-1421Crossref PubMed Scopus (43) Google Scholar), and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell (8Ferrari D. Los M. Bauer M.K.A. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (241) Google Scholar, 9Humphreys B.D. Rice J. Kertesy S.B. Dubyak G.R. J. Biol. Chem. 2000; 275: 26792-26798Abstract Full Text Full Text PDF PubMed Google Scholar). P2X7 activation also leads to extensive membrane blebbing (10Virginio C. MacKenzie A. North R.A. Surprenant A. J. Physiol. (Lond.). 1999; 519: 335-346Crossref Scopus (323) Google Scholar), which is a typical morphological feature of the apoptotic process. P2X7receptors have two transmembrane domains with intracellular amino and carboxyl termini, and the P2X7 receptor differs from other members of the P2X receptor family in having a long carboxyl terminus of 240 amino acids from the inner membrane face (11Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. J. Biol. Chem. 1997; 272: 5482-5486Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). This long carboxyl terminus is necessary for the permeability properties of the P2X7 receptor because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). P2X7 has an oligomeric structure in the membrane based on trimeric or larger complexes of identical subunits (13Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (481) Google Scholar, 14Kim M. Spelta V. Sim J. North R.A. Surprenant A. J. Biol. Chem. 2001; 276: 23262-23267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and there is evidence that P2X7 interacts with a number of structural and adhesion proteins in a complex at the cell surface (15Kim M. Jiang L.-H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar). Phosphorylation of a tyrosine at amino acid 343 of the P2X7primary structure has been proposed as being important for maintaining the full activity of the P2X7 channel (15Kim M. Jiang L.-H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar).A number of regulatory domains or motifs have been identified in the intracellular carboxyl tail based on homology with other proteins. These include a potential Src homology 3 binding domain (amino acids 450–456) and an ankyrin repeat motif (amino acids 494–508) (16Denlinger L.C. Fisette L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. J. Immunol. 2001; 167: 1871-1876Crossref PubMed Scopus (158) Google Scholar). Ankyrin repeats have been shown to play a major structural role in protein anchoring to the membrane or cytoskeleton, protein folding, and protein-protein interaction (17Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 18Rubstov A.M. Lopina O.D. FEBS Lett. 2000; 482: 1-5Crossref PubMed Scopus (107) Google Scholar). In addition, a lipopolysaccharide (LPS)1binding motif with homology to the LPS-binding protein of plasma (16Denlinger L.C. Fisette L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. J. Immunol. 2001; 167: 1871-1876Crossref PubMed Scopus (158) Google Scholar) has been proposed between amino acids 573 and 590, whereas we have shown that a region within this motif (amino acids 551–581) is necessary for the surface expression of P2X7 (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Upstream from this motif we have identified a single nucleotide polymorphism (1513A→C), present in around 207 of the population, which changes Glu to Ala at amino acid 496 and which leads to loss of function of the receptor (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Surface expression of P2X7 on lymphocytes was not affected by the Glu-496 to Ala polymorphism, suggesting that the loss of function resulted from impaired protein-protein interactions in the P2X7 complex at the cell membrane rather than from trafficking to the surface (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). In this study, we report the functional effects of a second polymorphism of the humanP2X7 gene (thymine to adenine at position 1729 of cDNA) that is associated with loss of function of the P2X7receptor because of failure of its trafficking to the cell surface. This polymorphism changes Ile to Asn at amino acid 568, which localizes this residue as being within a trafficking motif in the carboxyl tail of the receptor.DISCUSSIONThe present study has identified a single nucleotide polymorphism that leads to a loss of function of P2X7because of a trafficking defect in this receptor. This polymorphism was at position 1729T→A of the cDNA that changes isoleucine to asparagine at amino acid position 568. Three of 85 normal subjects were found to be heterozygous for this polymorphic variation, and all three had half-normal expression of the P2X7 on the cell surface and reduction of P2X7 function to ∼257 of normal. Transfection experiments provided confirmation that the Ile-568 to Asn-mutated P2X7 fails to traffic to the cell surface. HEK-293 cells transfected with this mutated P2X7 showed neither surface expression nor function despite plentiful intracellular synthesis of this receptor (Figs. 4 and 5). In other experiments, we measured the physiological properties of the Ile-568 to Asn-mutated P2X7 in Xenopus oocytes and found channel currents in oocytes that were identical to those with the wild type P2X7 construct. 2M. L. Smart, unpublished observations. This result shows that the Ile-568 to Asn mutation does not alter the function of the P2X7 channel, although this result shows differences in trafficking between the mammalian and amphibian expression systems. A number of functional domains have been proposed in the long carboxyl terminus of P2X7 based on protein sequence homology. The most distal of these, the "LPS-binding domain" from amino acids 573 to 590, has homology with the LPS-binding protein of plasma and has been suggested to interact directly with internalized LPS (16Denlinger L.C. Fisette L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. J. Immunol. 2001; 167: 1871-1876Crossref PubMed Scopus (158) Google Scholar). Using a series of truncated mutants of rat P2X7, we have identified previously a region between residues 551 and 581 that is both highly conserved and necessary for the surface expression of this receptor (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Mutation of the conserved residues at Cys-572, Arg-574, and Phe-581 abolished both cell surface expression and receptor function (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), suggesting that the trafficking domain of P2X7 overlaps the putative LPS-binding domain. The present data add the conserved Ile-568 to the other residues within this trafficking domain required for cell surface expression of P2X7. Whether this domain binds directly to phospholipid or to one of the many protein partners in the P2X7 membrane complex (15Kim M. Jiang L.-H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar) is uncertain. However, this domain includes a conserved cysteine, palmitoylation of which is required for cell surface expression in a number of other receptors such as CCR5 (28Blanpain C. Wittamer V. Vanderwinden J.M. Boom A. Renneboog B. Lee B. Le Poul E. El Asmar L. Govaerts C. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 2001; 276: 23795-23804Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Recently, P2X4 but not P2X2receptors have been shown to undergo constitutive endocytosis in neurons (29Bobanovic L.K. Royle S.J. Murrell-Lagnado R.D. J. Neurosci. 2002; 22: 4814-4824Crossref PubMed Google Scholar). Whether P2X7 also undergoes constitutive endocytosis is unknown, but the lack of 1729T→A-mutated P2X7 expressed on the surface of HEK-293 cells may be because of the rapid endocytosis of the receptor after initial trafficking to the cell surface. This, however, seems unlikely, as we failed to detect mutant P2X7 on the surface of HEK-293 cells using either flow cytometry or confocal microscopy.Wide variations in the function of the P2X7 receptor have been observed in lymphocytes both from normal subjects (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) and patients with CLL (30Wiley J.S. Dao-Ung L.P. Gu B.J. Sluyter R. Shemon A.N. Li C. Taper J. Gallo J. Manoharan A. Lancet. 2002; 359: 1114-1119Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Some but not all of this variability is a result of a single nucleotide polymorphism (1513A→C) that changes Glu-496 to Ala and leads to loss of function without affecting surface expression of the receptor in lymphocytes (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The trafficking-defective polymorphism identified in the present study, Ile-568 to Asn, contributes to this variability in P2X7function but with a lower heterozygote prevalence of ∼47 compared with ∼207 for the more common Glu-496 to Ala variation. Collectively, however, these two genotypes still do not account for all individuals with low or absent P2X7 function (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 30Wiley J.S. Dao-Ung L.P. Gu B.J. Sluyter R. Shemon A.N. Li C. Taper J. Gallo J. Manoharan A. Lancet. 2002; 359: 1114-1119Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The coding region within exon 13 of the P2X7 gene is highly polymorphic with five single nucleotide polymorphisms identified within a 0.5-kb stretch of cDNA, 3C. Li, R. Sluyter, B. J. Gu, and J. S. Wiley, unpublished observations. and it is likely that other loss-of-function polymorphisms exist in the P2X7gene, both within the coding region and in the upstream promoter of this gene (31Li C.M. Campbell S.J. Kumararatne D.S. Hill A.V.S. Lammas D.A. FEBS Lett. 2002; 531: 127-131Crossref PubMed Scopus (19) Google Scholar). Recently a polymorphism affecting the function of the mouse P2X7 receptor has been recognized in inbred strains of mice. Although most strains of mice carried Pro-451 with good P2X7 function, C57BL and DBA strains possessed a Leu-451 allele with lower P2X7 function (32Adriouch S. Dox C. Welge V. Seman M. Koch-Nolte F. Haag F. J. Immunol. 2002; 169: 4108-4112Crossref PubMed Scopus (169) Google Scholar). Whether the recognition or docking sequence around residue 451 lies within a domain interacting with a Src homology 3-binding protein is not known. We have surveyed 99 human base sequences coding for residue 451 but have found no deviations from the wild type sequence. 4C. Li and J. S. Wiley, unpublished observations. An increasing number of channelopathies have been associated with human disease. Thus hypokalemic periodic paralysis and familial hemiplegic migraine result from missense or point mutations in a voltage-sensitive calcium channel (33Jurkat-Rott K. Lehmann-Horn F. Elbaz A. Heine R. Gregg R.G. Hogan K. Powers P.A. Lapie P. Vale-Santos J.E. Weissenbach J. Hum. Mol. Genet. 1994; 3: 1415-1419Crossref PubMed Scopus (275) Google Scholar, 34Ophoff R.A. Terwindt G.M. Vergouwe M.N. van Eijk R. Oefner P.J. Hoffman S.M. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2097) Google Scholar). Mutations in sodium and potassium channels also can cause neurological disease with ataxic or epileptic phenotypes (34Ophoff R.A. Terwindt G.M. Vergouwe M.N. van Eijk R. Oefner P.J. Hoffman S.M. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2097) Google Scholar, 35Biervert C. Schroeder B.C. Kubisch C. Berkovic S.F. Propping P. Jentsch T.J. Steinlein O.K. Science. 1998; 279: 403-406Crossref PubMed Scopus (925) Google Scholar, 36Wallace R.H. Wang D.W. Singh R. Scheffer I.E. George A.L. Phillips H.A. Saar K. Reis A. Johnson E.W. Sutherland G.R. Berkovic S.F. Mulley J.C. Nat. Genet. 1998; 19: 366-370Crossref PubMed Scopus (94) Google Scholar). There is increasing interest in channelopathies that affect cells of the immune system and the impact of reduced or absent P2X7 channel function on human susceptibility to infectious diseases. P2X7 receptor expression is up-regulated on differentiation of monocytes to macrophages (27Hickman S.E. El Khoury J. Greenberg S. Schieren I. Silverstein S.C. Blood. 1994; 84: 2452-2456Crossref PubMed Google Scholar), where its activation is necessary both for the ATP-induced killing of ingested mycobacterial species and for the subsequent apoptotic death of this phagocytic cell (37Lammas D.A. Stober C. Harvey C.J. Kendrick N. Panchalingam S. Kumararatne D.S. Immunity. 1997; 7: 433-444Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 38Kusner D.J. Adams J. J. Immunol. 2000; 164: 379-388Crossref PubMed Scopus (147) Google Scholar, 39Fairbairn I.P. Stober C.B. Kumararatne D.S. Lammas D.A. J. Immunol. 2001; 167: 3300-3307Crossref PubMed Scopus (207) Google Scholar). Whether Asn-568 (1729A allele) as well as Ala-496 (1513C allele) contribute to the genetic susceptibility to tuberculous infection is uncertain, although macrophages from subjects heterozygous for Asn-568 failed to up-regulate P2X7 function to the same extent as wild type subjects (Fig. 6). Although a mouse strain of P2X7-null genotype has been developed, there is no distinctive adverse phenotype of this animal (40Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. J. Biol. Chem. 2001; 276: 125-132Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar, 41Labasi J.M. Petrushova N. Donovan C. McCurdy S. Lira P. Payette M.M. Brissette W. Wicks J.R. Audoly L. Gabel C.A. J. Immunol. 2002; 168: 6436-6445Crossref PubMed Scopus (436) Google Scholar). However, the severity of inflammatory arthritis induced by anti-collagen antibody was markedly attenuated in these animals (41Labasi J.M. Petrushova N. Donovan C. McCurdy S. Lira P. Payette M.M. Brissette W. Wicks J.R. Audoly L. Gabel C.A. J. Immunol. 2002; 168: 6436-6445Crossref PubMed Scopus (436) Google Scholar). It seems likely that the P2X7 receptor and its polymorphic variants will be central in our understanding of certain inflammatory and infectious diseases. The purinergic P2X7 receptor is a ligand-gated channel, selective for cationic permeants, which has a wide distribution including cells of the immune and hemopoietic system (1Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Crossref PubMed Scopus (616) Google Scholar,2North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2438) Google Scholar). Activation of this receptor by brief exposure to extracellular ATP opens a channel that allows Ca2+ and Na+ influx and K+ efflux and that initiates a cascade of intracellular downstream events. These include the stimulation of phospholipase D (3El-Moatassim C. Dubyak G.R. J. Biol. Chem. 1993; 268: 15571-15578Abstract Full Text PDF PubMed Google Scholar,4Gargett C.E. Cornish E.J. Wiley J.S. Biochem. J. 1996; 313: 529-535Crossref PubMed Scopus (65) Google Scholar), the activation of membrane metalloproteases (5Jamieson G.P. Snook M.B. Thurlow P.J. Wiley J.S. J. Cell. Physiol. 1996; 166: 637-642Crossref PubMed Scopus (84) Google Scholar, 6Gu B. Bendall L.J. Wiley J.S. Blood. 1998; 92: 946-951Crossref PubMed Google Scholar, 7Sluyter R. Wiley J.S. Int. Immunol. 2002; 14: 1415-1421Crossref PubMed Scopus (43) Google Scholar), and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell (8Ferrari D. Los M. Bauer M.K.A. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (241) Google Scholar, 9Humphreys B.D. Rice J. Kertesy S.B. Dubyak G.R. J. Biol. Chem. 2000; 275: 26792-26798Abstract Full Text Full Text PDF PubMed Google Scholar). P2X7 activation also leads to extensive membrane blebbing (10Virginio C. MacKenzie A. North R.A. Surprenant A. J. Physiol. (Lond.). 1999; 519: 335-346Crossref Scopus (323) Google Scholar), which is a typical morphological feature of the apoptotic process. P2X7receptors have two transmembrane domains with intracellular amino and carboxyl termini, and the P2X7 receptor differs from other members of the P2X receptor family in having a long carboxyl terminus of 240 amino acids from the inner membrane face (11Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. J. Biol. Chem. 1997; 272: 5482-5486Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). This long carboxyl terminus is necessary for the permeability properties of the P2X7 receptor because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1487) Google Scholar). P2X7 has an oligomeric structure in the membrane based on trimeric or larger complexes of identical subunits (13Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (481) Google Scholar, 14Kim M. Spelta V. Sim J. North R.A. Surprenant A. J. Biol. Chem. 2001; 276: 23262-23267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and there is evidence that P2X7 interacts with a number of structural and adhesion proteins in a complex at the cell surface (15Kim M. Jiang L.-H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar). Phosphorylation of a tyrosine at amino acid 343 of the P2X7primary structure has been proposed as being important for maintaining the full activity of the P2X7 channel (15Kim M. Jiang L.-H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar). A number of regulatory domains or motifs have been identified in the intracellular carboxyl tail based on homology with other proteins. These include a potential Src homology 3 binding domain (amino acids 450–456) and an ankyrin repeat motif (amino acids 494–508) (16Denlinger L.C. Fisette L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. J. Immunol. 2001; 167: 1871-1876Crossref PubMed Scopus (158) Google Scholar). Ankyrin repeats have been shown to play a major structural role in protein anchoring to the membrane or cytoskeleton, protein folding, and protein-protein interaction (17Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 18Rubstov A.M. Lopina O.D. FEBS Lett. 2000; 482: 1-5Crossref PubMed Scopus (107) Google Scholar). In addition, a lipopolysaccharide (LPS)1binding motif with homology to the LPS-binding protein of plasma (16Denlinger L.C. Fisette L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. J. Immunol. 2001; 167: 1871-1876Crossref PubMed Scopus (158) Google Scholar) has been proposed between amino acids 573 and 590, whereas we have shown that a region within this motif (amino acids 551–581) is necessary for the surface expression of P2X7 (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Upstream from this motif we have identified a single nucleotide polymorphism (1513A→C), present in around 207 of the population, which changes Glu to Ala at amino acid 496 and which leads to loss of function of the receptor (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Surface expression of P2X7 on lymphocytes was not affected by the Glu-496 to Ala polymorphism, suggesting that the loss of function resulted from impaired protein-protein interactions in the P2X7 complex at the cell membrane rather than from trafficking to the surface (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). In this study, we report the functional effects of a second polymorphism of the humanP2X7 gene (thymine to adenine at position 1729 of cDNA) that is associated with loss of function of the P2X7receptor because of failure of its trafficking to the cell surface. This polymorphism changes Ile to Asn at amino acid 568, which localizes this residue as being within a trafficking motif in the carboxyl tail of the receptor. DISCUSSIONThe present study has identified a single nucleotide polymorphism that leads to a loss of function of P2X7because of a trafficking defect in this receptor. This polymorphism was at position 1729T→A of the cDNA that changes isoleucine to asparagine at amino acid position 568. Three of 85 normal subjects were found to be heterozygous for this polymorphic variation, and all three had half-normal expression of the P2X7 on the cell surface and reduction of P2X7 function to ∼257 of normal. Transfection experiments provided confirmation that the Ile-568 to Asn-mutated P2X7 fails to traffic to the cell surface. HEK-293 cells transfected with this mutated P2X7 showed neither surface expression nor function despite plentiful intracellular synthesis of this receptor (Figs. 4 and 5). In other experiments, we measured the physiological properties of the Ile-568 to Asn-mutated P2X7 in Xenopus oocytes and found channel currents in oocytes that were identical to those with the wild type P2X7 construct. 2M. L. Smart, unpublished observations. This result shows that the Ile-568 to Asn mutation does not alter the function of the P2X7 channel, although this result shows differences in trafficking between the mammalian and amphibian expression systems. A number of functional domains have been proposed in the long carboxyl terminus of P2X7 based on protein sequence homology. The most distal of these, the "LPS-binding domain" from amino acids 573 to 590, has homology with the LPS-binding protein of plasma and has been suggested to interact directly with internalized LPS (16Denlinger L.C. Fisette L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. J. Immunol. 2001; 167: 1871-1876Crossref PubMed Scopus (158) Google Scholar). Using a series of truncated mutants of rat P2X7, we have identified previously a region between residues 551 and 581 that is both highly conserved and necessary for the surface expression of this receptor (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Mutation of the conserved residues at Cys-572, Arg-574, and Phe-581 abolished both cell surface expression and receptor function (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), suggesting that the trafficking domain of P2X7 overlaps the putative LPS-binding domain. The present data add the conserved Ile-568 to the other residues within this trafficking domain required for cell surface expression of P2X7. Whether this domain binds directly to phospholipid or to one of the many protein partners in the P2X7 membrane complex (15Kim M. Jiang L.-H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar) is uncertain. However, this domain includes a conserved cysteine, palmitoylation of which is required for cell surface expression in a number of other receptors such as CCR5 (28Blanpain C. Wittamer V. Vanderwinden J.M. Boom A. Renneboog B. Lee B. Le Poul E. El Asmar L. Govaerts C. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 2001; 276: 23795-23804Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Recently, P2X4 but not P2X2receptors have been shown to undergo constitutive endocytosis in neurons (29Bobanovic L.K. Royle S.J. Murrell-Lagnado R.D. J. Neurosci. 2002; 22: 4814-4824Crossref PubMed Google Scholar). Whether P2X7 also undergoes constitutive endocytosis is unknown, but the lack of 1729T→A-mutated P2X7 expressed on the surface of HEK-293 cells may be because of the rapid endocytosis of the receptor after initial trafficking to the cell surface. This, however, seems unlikely, as we failed to detect mutant P2X7 on the surface of HEK-293 cells using either flow cytometry or confocal microscopy.Wide variations in the function of the P2X7 receptor have been observed in lymphocytes both from normal subjects (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) and patients with CLL (30Wiley J.S. Dao-Ung L.P. Gu B.J. Sluyter R. Shemon A.N. Li C. Taper J. Gallo J. Manoharan A. Lancet. 2002; 359: 1114-1119Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Some but not all of this variability is a result of a single nucleotide polymorphism (1513A→C) that changes Glu-496 to Ala and leads to loss of function without affecting surface expression of the receptor in lymphocytes (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The trafficking-defective polymorphism identified in the present study, Ile-568 to Asn, contributes to this variability in P2X7function but with a lower heterozygote prevalence of ∼47 compared with ∼207 for the more common Glu-496 to Ala variation. Collectively, however, these two genotypes still do not account for all individuals with low or absent P2X7 function (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 30Wiley J.S. Dao-Ung L.P. Gu B.J. Sluyter R. Shemon A.N. Li C. Taper J. Gallo J. Manoharan A. Lancet. 2002; 359: 1114-1119Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The coding region within exon 13 of the P2X7 gene is highly polymorphic with five single nucleotide polymorphisms identified within a 0.5-kb stretch of cDNA, 3C. Li, R. Sluyter, B. J. Gu, and J. S. Wiley, unpublished observations. and it is likely that other loss-of-function polymorphisms exist in the P2X7gene, both within the coding region and in the upstream promoter of this gene (31Li C.M. Campbell S.J. Kumararatne D.S. Hill A.V.S. Lammas D.A. FEBS Lett. 2002; 531: 127-131Crossref PubMed Scopus (19) Google Scholar). Recently a polymorphism affecting the function of the mouse P2X7 receptor has been recognized in inbred strains of mice. Although most strains of mice carried Pro-451 with good P2X7 function, C57BL and DBA strains possessed a Leu-451 allele with lower P2X7 function (32Adriouch S. Dox C. Welge V. Seman M. Koch-Nolte F. Haag F. J. Immunol. 2002; 169: 4108-4112Crossref PubMed Scopus (169) Google Scholar). Whether the recognition or docking sequence around residue 451 lies within a domain interacting with a Src homology 3-binding protein is not known. We have surveyed 99 human base sequences coding for residue 451 but have found no deviations from the wild type sequence. 4C. Li and J. S. Wiley, unpublished observations. An increasing number of channelopathies have been associated with human disease. Thus hypokalemic periodic paralysis and familial hemiplegic migraine result from missense or point mutations in a voltage-sensitive calcium channel (33Jurkat-Rott K. Lehmann-Horn F. Elbaz A. Heine R. Gregg R.G. Hogan K. Powers P.A. Lapie P. Vale-Santos J.E. Weissenbach J. Hum. Mol. Genet. 1994; 3: 1415-1419Crossref PubMed Scopus (275) Google Scholar, 34Ophoff R.A. Terwindt G.M. Vergouwe M.N. van Eijk R. Oefner P.J. Hoffman S.M. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2097) Google Scholar). Mutations in sodium and potassium channels also can cause neurological disease with ataxic or epileptic phenotypes (34Ophoff R.A. Terwindt G.M. Vergouwe M.N. van Eijk R. Oefner P.J. Hoffman S.M. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2097) Google Scholar, 35Biervert C. Schroeder B.C. Kubisch C. Berkovic S.F. Propping P. Jentsch T.J. Steinlein O.K. Science. 1998; 279: 403-406Crossref PubMed Scopus (925) Google Scholar, 36Wallace R.H. Wang D.W. Singh R. Scheffer I.E. George A.L. Phillips H.A. Saar K. Reis A. Johnson E.W. Sutherland G.R. Berkovic S.F. Mulley J.C. Nat. Genet. 1998; 19: 366-370Crossref PubMed Scopus (94) Google Scholar). There is increasing interest in channelopathies that affect cells of the immune system and the impact of reduced or absent P2X7 channel function on human susceptibility to infectious diseases. P2X7 receptor expression is up-regulated on differentiation of monocytes to macrophages (27Hickman S.E. El Khoury J. Greenberg S. Schieren I. Silverstein S.C. Blood. 1994; 84: 2452-2456Crossref PubMed Google Scholar), where its activation is necessary both for the ATP-induced killing of ingested mycobacterial species and for the subsequent apoptotic death of this phagocytic cell (37Lammas D.A. Stober C. Harvey C.J. Kendrick N. Panchalingam S. Kumararatne D.S. Immunity. 1997; 7: 433-444Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 38Kusner D.J. Adams J. J. Immunol. 2000; 164: 379-388Crossref PubMed Scopus (147) Google Scholar, 39Fairbairn I.P. Stober C.B. Kumararatne D.S. Lammas D.A. J. Immunol. 2001; 167: 3300-3307Crossref PubMed Scopus (207) Google Scholar). Whether Asn-568 (1729A allele) as well as Ala-496 (1513C allele) contribute to the genetic susceptibility to tuberculous infection is uncertain, although macrophages from subjects heterozygous for Asn-568 failed to up-regulate P2X7 function to the same extent as wild type subjects (Fig. 6). Although a mouse strain of P2X7-null genotype has been developed, there is no distinctive adverse phenotype of this animal (40Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. J. Biol. Chem. 2001; 276: 125-132Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar, 41Labasi J.M. Petrushova N. Donovan C. McCurdy S. Lira P. Payette M.M. Brissette W. Wicks J.R. Audoly L. Gabel C.A. J. Immunol. 2002; 168: 6436-6445Crossref PubMed Scopus (436) Google Scholar). However, the severity of inflammatory arthritis induced by anti-collagen antibody was markedly attenuated in these animals (41Labasi J.M. Petrushova N. Donovan C. McCurdy S. Lira P. Payette M.M. Brissette W. Wicks J.R. Audoly L. Gabel C.A. J. Immunol. 2002; 168: 6436-6445Crossref PubMed Scopus (436) Google Scholar). It seems likely that the P2X7 receptor and its polymorphic variants will be central in our understanding of certain inflammatory and infectious diseases. The present study has identified a single nucleotide polymorphism that leads to a loss of function of P2X7because of a trafficking defect in this receptor. This polymorphism was at position 1729T→A of the cDNA that changes isoleucine to asparagine at amino acid position 568. Three of 85 normal subjects were found to be heterozygous for this polymorphic variation, and all three had half-normal expression of the P2X7 on the cell surface and reduction of P2X7 function to ∼257 of normal. Transfection experiments provided confirmation that the Ile-568 to Asn-mutated P2X7 fails to traffic to the cell surface. HEK-293 cells transfected with this mutated P2X7 showed neither surface expression nor function despite plentiful intracellular synthesis of this receptor (Figs. 4 and 5). In other experiments, we measured the physiological properties of the Ile-568 to Asn-mutated P2X7 in Xenopus oocytes and found channel currents in oocytes that were identical to those with the wild type P2X7 construct. 2M. L. Smart, unpublished observations. This result shows that the Ile-568 to Asn mutation does not alter the function of the P2X7 channel, although this result shows differences in trafficking between the mammalian and amphibian expression systems. A number of functional domains have been proposed in the long carboxyl terminus of P2X7 based on protein sequence homology. The most distal of these, the "LPS-binding domain" from amino acids 573 to 590, has homology with the LPS-binding protein of plasma and has been suggested to interact directly with internalized LPS (16Denlinger L.C. Fisette L. Sommer J.A. Watters J.J. Prabhu U. Dubyak G.R. Proctor R.A. Bertics P.J. J. Immunol. 2001; 167: 1871-1876Crossref PubMed Scopus (158) Google Scholar). Using a series of truncated mutants of rat P2X7, we have identified previously a region between residues 551 and 581 that is both highly conserved and necessary for the surface expression of this receptor (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Mutation of the conserved residues at Cys-572, Arg-574, and Phe-581 abolished both cell surface expression and receptor function (19Smart M.L. Gu B. Panchel R.G. Wiley J. Cromer B. Williams D.A. Petrou S. J. Biol. Chem. 2003; 278: 8853-8860Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), suggesting that the trafficking domain of P2X7 overlaps the putative LPS-binding domain. The present data add the conserved Ile-568 to the other residues within this trafficking domain required for cell surface expression of P2X7. Whether this domain binds directly to phospholipid or to one of the many protein partners in the P2X7 membrane complex (15Kim M. Jiang L.-H. Wilson H.L. North R.A. Surprenant A. EMBO J. 2001; 20: 6347-6358Crossref PubMed Scopus (334) Google Scholar) is uncertain. However, this domain includes a conserved cysteine, palmitoylation of which is required for cell surface expression in a number of other receptors such as CCR5 (28Blanpain C. Wittamer V. Vanderwinden J.M. Boom A. Renneboog B. Lee B. Le Poul E. El Asmar L. Govaerts C. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 2001; 276: 23795-23804Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Recently, P2X4 but not P2X2receptors have been shown to undergo constitutive endocytosis in neurons (29Bobanovic L.K. Royle S.J. Murrell-Lagnado R.D. J. Neurosci. 2002; 22: 4814-4824Crossref PubMed Google Scholar). Whether P2X7 also undergoes constitutive endocytosis is unknown, but the lack of 1729T→A-mutated P2X7 expressed on the surface of HEK-293 cells may be because of the rapid endocytosis of the receptor after initial trafficking to the cell surface. This, however, seems unlikely, as we failed to detect mutant P2X7 on the surface of HEK-293 cells using either flow cytometry or confocal microscopy. Wide variations in the function of the P2X7 receptor have been observed in lymphocytes both from normal subjects (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) and patients with CLL (30Wiley J.S. Dao-Ung L.P. Gu B.J. Sluyter R. Shemon A.N. Li C. Taper J. Gallo J. Manoharan A. Lancet. 2002; 359: 1114-1119Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Some but not all of this variability is a result of a single nucleotide polymorphism (1513A→C) that changes Glu-496 to Ala and leads to loss of function without affecting surface expression of the receptor in lymphocytes (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). The trafficking-defective polymorphism identified in the present study, Ile-568 to Asn, contributes to this variability in P2X7function but with a lower heterozygote prevalence of ∼47 compared with ∼207 for the more common Glu-496 to Ala variation. Collectively, however, these two genotypes still do not account for all individuals with low or absent P2X7 function (20Gu B.J. Zhang W.Y. Worthington R.A. Sluyter R. Dao-Ung P. Petrou S. Barden J.A. Wiley J.S. J. Biol. Chem. 2001; 276: 11135-11142Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 30Wiley J.S. Dao-Ung L.P. Gu B.J. Sluyter R. Shemon A.N. Li C. Taper J. Gallo J. Manoharan A. Lancet. 2002; 359: 1114-1119Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The coding region within exon 13 of the P2X7 gene is highly polymorphic with five single nucleotide polymorphisms identified within a 0.5-kb stretch of cDNA, 3C. Li, R. Sluyter, B. J. Gu, and J. S. Wiley, unpublished observations. and it is likely that other loss-of-function polymorphisms exist in the P2X7gene, both within the coding region and in the upstream promoter of this gene (31Li C.M. Campbell S.J. Kumararatne D.S. Hill A.V.S. Lammas D.A. FEBS Lett. 2002; 531: 127-131Crossref PubMed Scopus (19) Google Scholar). Recently a polymorphism affecting the function of the mouse P2X7 receptor has been recognized in inbred strains of mice. Although most strains of mice carried Pro-451 with good P2X7 function, C57BL and DBA strains possessed a Leu-451 allele with lower P2X7 function (32Adriouch S. Dox C. Welge V. Seman M. Koch-Nolte F. Haag F. J. Immunol. 2002; 169: 4108-4112Crossref PubMed Scopus (169) Google Scholar). Whether the recognition or docking sequence around residue 451 lies within a domain interacting with a Src homology 3-binding protein is not known. We have surveyed 99 human base sequences coding for residue 451 but have found no deviations from the wild type sequence. 4C. Li and J. S. Wiley, unpublished observations. An increasing number of channelopathies have been associated with human disease. Thus hypokalemic periodic paralysis and familial hemiplegic migraine result from missense or point mutations in a voltage-sensitive calcium channel (33Jurkat-Rott K. Lehmann-Horn F. Elbaz A. Heine R. Gregg R.G. Hogan K. Powers P.A. Lapie P. Vale-Santos J.E. Weissenbach J. Hum. Mol. Genet. 1994; 3: 1415-1419Crossref PubMed Scopus (275) Google Scholar, 34Ophoff R.A. Terwindt G.M. Vergouwe M.N. van Eijk R. Oefner P.J. Hoffman S.M. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2097) Google Scholar). Mutations in sodium and potassium channels also can cause neurological disease with ataxic or epileptic phenotypes (34Ophoff R.A. Terwindt G.M. Vergouwe M.N. van Eijk R. Oefner P.J. Hoffman S.M. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2097) Google Scholar, 35Biervert C. Schroeder B.C. Kubisch C. Berkovic S.F. Propping P. Jentsch T.J. Steinlein O.K. Science. 1998; 279: 403-406Crossref PubMed Scopus (925) Google Scholar, 36Wallace R.H. Wang D.W. Singh R. Scheffer I.E. George A.L. Phillips H.A. Saar K. Reis A. Johnson E.W. Sutherland G.R. Berkovic S.F. Mulley J.C. Nat. Genet. 1998; 19: 366-370Crossref PubMed Scopus (94) Google Scholar). There is increasing interest in channelopathies that affect cells of the immune system and the impact of reduced or absent P2X7 channel function on human susceptibility to infectious diseases. P2X7 receptor expression is up-regulated on differentiation of monocytes to macrophages (27Hickman S.E. El Khoury J. Greenberg S. Schieren I. Silverstein S.C. Blood. 1994; 84: 2452-2456Crossref PubMed Google Scholar), where its activation is necessary both for the ATP-induced killing of ingested mycobacterial species and for the subsequent apoptotic death of this phagocytic cell (37Lammas D.A. Stober C. Harvey C.J. Kendrick N. Panchalingam S. Kumararatne D.S. Immunity. 1997; 7: 433-444Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 38Kusner D.J. Adams J. J. Immunol. 2000; 164: 379-388Crossref PubMed Scopus (147) Google Scholar, 39Fairbairn I.P. Stober C.B. Kumararatne D.S. Lammas D.A. J. Immunol. 2001; 167: 3300-3307Crossref PubMed Scopus (207) Google Scholar). Whether Asn-568 (1729A allele) as well as Ala-496 (1513C allele) contribute to the genetic susceptibility to tuberculous infection is uncertain, although macrophages from subjects heterozygous for Asn-568 failed to up-regulate P2X7 function to the same extent as wild type subjects (Fig. 6). Although a mouse strain of P2X7-null genotype has been developed, there is no distinctive adverse phenotype of this animal (40Solle M. Labasi J. Perregaux D.G. Stam E. Petrushova N. Koller B.H. Griffiths R.J. Gabel C.A. J. Biol. Chem. 2001; 276: 125-132Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar, 41Labasi J.M. Petrushova N. Donovan C. McCurdy S. Lira P. Payette M.M. Brissette W. Wicks J.R. Audoly L. Gabel C.A. J. Immunol. 2002; 168: 6436-6445Crossref PubMed Scopus (436) Google Scholar). However, the severity of inflammatory arthritis induced by anti-collagen antibody was markedly attenuated in these animals (41Labasi J.M. Petrushova N. Donovan C. McCurdy S. Lira P. Payette M.M. Brissette W. Wicks J.R. Audoly L. Gabel C.A. J. Immunol. 2002; 168: 6436-6445Crossref PubMed Scopus (436) Google Scholar). It seems likely that the P2X7 receptor and its polymorphic variants will be central in our understanding of certain inflammatory and infectious diseases. We thank Dr. Diane Williams for blood collection; Prof. Graeme Stewart, Dr. David Booth, and Maria Ban for helpful discussions; Shelley Spicer for typing this manuscript; and Kristen Skarratt for critically reviewing the manuscript.