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The P2X 7 receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X 7 is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X 7 . In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A3 C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X 7 on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X 7 function that was half that of germline P2X 7 . Results of transfection experiments showed that the mutant P2X 7 receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X 7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-␥, which up-regulated mutant P2X 7 and partially restored its function. P2X 7 -mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X 7 compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X 7 receptor.Purinergic P2X 7 receptors are ligand-gated cation channels, present on cells of the immune and hemopoietic system, that have been shown to mediate the ATP-induced apoptotic death of monocytes (1), macrophages (2), and lymphocytes (3, 4). The P2X 7 receptor family has two transmembrane domains with intracellular amino and carboxyl termini and an oligomeric structure in the plasma membrane based on trimeric or larger complexes of identical subunits (5). Moreover, the P2X7 receptor does not appear to form heteropolymers with other P2X subtypes (6). The genes for both the rat and human P2X 7 receptors have now been cloned and show extensive homology (30 -40%) with the other members of the P2X receptor family, although P2X 7 differs in having a long carboxyl terminus of 240 amino acids from the inner membrane face (7). The genomic structure of P2X 7 consists of 13 exons, with exon 12 and exon 13 coding for the C-terminal tail of this molecule. There is strong evidence that 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 YoPro-1 (8). Studies of P2X 7 of macrophages or lymphocytes as well as of human embryonic kidney cells (HEK-293) expressing the cDNA for P2X 7 have shown features that are most unusual for a channel. These include the slow further dilatation following channel opening (9) and the activation of various proteases including membrane metalloproteases (10) and intracellular caspases (2, 11). The fully ...

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.