The P2X 7 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 P2X 7 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. P2X 7 function was measured by ATPinduced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into various lymphocyte subsets and was decreased to values of ϳ25% of normal. The expression of the P2X 7 receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X 7 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-␥ up-regulated P2X 7 function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X 7 receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.The purinergic P2X 7 receptor is a ligand-gated channel, selective for cationic permeants, which has a wide distribution including cells of the immune and hemopoietic system (1, 2). Activation of this receptor by brief exposure to extracellular ATP opens a channel that allows Ca 2ϩ and Na ϩ influx and K ϩ efflux and that initiates a cascade of intracellular downstream events. These include the stimulation of phospholipase D (3, 4), the activation of membrane metalloproteases (5-7), and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell (8, 9). P2X 7 activation also leads to extensive membrane blebbing (10), which is a typical morphological feature of the apoptotic process. P2X 7 receptors have two transmembrane domains with intracellular amino and carboxyl termini, and the P2X 7 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 (11). This long carboxyl terminus is necessary for the permeability properties of the P2X 7 receptor because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12). P2X 7 has an oligomeric structure in the membrane based on trimeric or larger complexes of identical subunits (13,14), and there is evidence that P2X 7 interacts with a number of structural and adhesion proteins in a complex at the cell surface (15). Phosphorylation of a tyrosine at amino acid 343 of the P2X 7 primary structure has been proposed as being important for maintaining the full activity of the P2X 7 channel (15). A number of regulatory d...

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.

The P2X 7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X 7 receptor have been observed, explained in part by loss-of-function polymorphisms that change Glu 496 to Ala (E496A) and Ile 568 to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg 307 (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X 7 function was measured by ATP-induced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X 7 carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X 7 and blocks P2X 7 function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X 7 function in cells heterozygous for the R307Q to a value 10 -40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X 7 , and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X 7 receptor, and since the affected Arg 307 is homologous to those amino acids essential for ATP binding to P2X 1 and P2X 2 , it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X 7 .In cells of the hemopoietic and immune systems, extracellular ATP can induce cytolysis of lymphocytes (1), monocytes/ macrophages (2), and dendritic cells (3). It is generally accepted that these cytolytic effects of ATP are mediated by the P2X 7 receptor, which is a ligand-gated cation channel activated by extracellular ATP and highly expressed on these cell types (4, 5 P2X receptors have an oligomeric structure in the plasma membrane based on trimeric or larger complexes of identical subunits (20,21). Moreover the values of Hill coefficients derived from the sigmoid ATP dose-response curves of the P2X 7 (P2Z) receptor are consistent with multiple ATP-binding sites in each P2X 7 trimer (8,22,23). All seven members of the P2X receptor family have two transmembrane domains with intracellular amino and carboxyl termini. Little is known of the conformation of the extracellular domain containing the ATPbinding site(s). An analysis of the P2X subtype sequence homology has shown that the two transmembrane domains M1 and M2 are separated by an extracellular sequence containing a cysteine-rich region (residues 110 -170) followed by a segment from Phe 188 -Val 321 that may form six antiparallel ␤-pleated sheets homologous with members of the class II aminoacyl-tRNA synthetases (24). The ATP-...