The P2X₇ 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 P2X₇ 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 P2X₇. In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A→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 P2X₇ 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 P2X₇ function that was half that of germline P2X₇. Results of transfection experiments showed that the mutant P2X₇ receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X₇ function was also seen on differentiation of fresh monocytes to macrophages with interferon-γ, which up-regulated mutant P2X₇ and partially restored its function. P2X₇-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X₇ compared with germline (8.6versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X₇receptor.

The importance of the cytosolic C-terminal region of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake, whereas those expressing the P2X7R truncated at position 582 give wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region between residues 551 and 581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues within this region could also abolish receptor function and cell surface expression, suggesting that this region may participate in regulating the surface expression of the pore-forming P2X7R.

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 wildtype 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‐1β 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.—Stokes, L., Fuller, S. J., Sluyter, R., Skarratt, K. K., Gu, B. J., Wiley, J. S. Two haplotypes of the P2X ₇ receptor containing the Ala‐348 to Thr polymorphism exhibit a gain‐of‐function effect and enhanced interleukin‐1β secretion. FASEB J . 24, 2916–2927 (2010). www.fasebj.org

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 ∼25% 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 50% 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 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 P2X₇ 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 P2X₇ polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C → G), which changes Thr³⁵⁷ to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X₇ 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 P2X₇ in either HEK-293 cells or Xenopus oocytes gave P2X₇ function of ∼50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X₇, restored P2X₇ 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 P2X₇ 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 P2X₇ receptor function.