A Glu-496 to Ala Polymorphism Leads to Loss of Function of the Human P2X7 Receptor

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 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. fluorescein isothiocyanate benzoylbenzoyl polymerase chain reaction monoclonal antibody human embryonic kidney cluster of differentiation Purinergic P2X7 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 (1Grahames C.B. Michel A.D. Chessell I.P. Humphrey P.P. Br. J. Pharmacol. 1999; 127: 1915-1921Crossref PubMed Scopus (127) Google Scholar), macrophages (2Humphreys 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), and lymphocytes (3Chused T.M. Apasov S. Sitkovsky M. J. Immunol. 1996; 157: 1371-1380PubMed Google Scholar, 4Peng L. Bradley C.J. Wiley J.S. Chin. Med. J. ( Engl. Ed. ). 1998; 78: 508-511Google Scholar). The P2X7receptor 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 (5Nicke 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). Moreover, the P2X7 receptor does not appear to form heteropolymers with other P2X subtypes (6Torres 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). The genes for both the rat and human P2X7 receptors have now been cloned and show extensive homology (30–40%) with the other members of the P2X receptor family, although P2X7 differs in having a long carboxyl terminus of 240 amino acids from the inner membrane face (7Rassendren F. Buell G.N. Virginio C. Collo G. North R.A. Surprenant A. J. Biol. Chem. 1997; 272: 5482-5486Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). The genomic structure of P2X7 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 P2X7 receptor, because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye YoPro-1 (8Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1545) Google Scholar). Studies of P2X7 of macrophages or lymphocytes as well as of human embryonic kidney cells (HEK-293) expressing the cDNA for P2X7 have shown features that are most unusual for a channel. These include the slow further dilatation following channel opening (9Wiley J.S. Gargett C.E. Zhang W. Snook M.B. Jamieson G.P. Am. J. Physiol. 1998; 275: C1224-C1231Crossref PubMed Google Scholar) and the activation of various proteases including membrane metalloproteases (10Gu B. Bendall L.J. Wiley J.S. Blood. 1998; 92: 946-951Crossref PubMed Google Scholar) and intracellular caspases (2Humphreys 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, 11Ferrari D. Los M. Bauer M.K. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (244) Google Scholar). The fully dilated state of the P2X7 pore accepts ethidium cation (314 Da) as a permeant, and because ethidium fluorescence is enhanced on binding to nucleic acids, the technique of flow cytometry allows a sensitive measurement of the initial rate of permeant uptake that is essentially unidirectional (9Wiley J.S. Gargett C.E. Zhang W. Snook M.B. Jamieson G.P. Am. J. Physiol. 1998; 275: C1224-C1231Crossref PubMed Google Scholar). In normal leukocytes a close correlation has been found between ATP-induced ethidium uptake and the surface expression of P2X7receptors measured by the binding of a fluorescein isothiocyanate (FITC)1-conjugated antibody to the extracellular domain of this receptor (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar). There is increasing evidence that a genetic factor plays a role in the functional phenotype of the P2X7 receptor. Thus, Lammaset al. (13Lammas 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 (351) Google Scholar) have shown that ATP-induced uptake of the dye lucifer yellow into monocytes was minimal in 2 of 19 normal donors, whereas our group has shown a lack of P2X7 function in both lymphocytes and monocytes in 3 of 12 patients with B-cell chronic lymphocytic leukemia despite strong expression of the P2X7 protein (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar). These results led us to search for nonfunctional P2X7 receptors in a large cohort of normal subjects and study its possible genetic basis. The results show that a single nucleotide polymorphism is present at low frequency in the Caucasian population and codes for a glutamic acid to alanine substitution at amino acid 496. Homozygosity for the polymorphism produces nonfunctional P2X7 protein, whereas the heterozygous state gives cells with half the function of cells with germline P2X7 protein. ATP, BzATP (2′, 3′-O-(4-benzoyl)benzoyl ATP), ethidium bromide, barium chloride, digitonin, d-glucose, bovine serum albumin, RPMI 1640 medium, poly-l-lysine, gentamicin, 7-amino-actinomycin D, and FluoroTag FITC conjugation kit were purchased from Sigma. Ficoll-Hypaque (density 1.077) and GFXTM PCR DNA and gel band purification kit were obtained from Amersham Pharmacia Biotech. FITC- and R-phycoerythrin-conjugated negative control antibodies, mouse anti-human CD3, CD14, CD16, and CD19 antibodies, and R-phycoerythrin-Cy5-conjugated mouse anti-human CD19 antibody were from Dako (Carpinteria, CA). Cy2-conjugated donkey anti-mouse IgG antibody was from Jackson ImmunoResearch (West Grove, PA). Hepes, LipofectAMINETM 2000 reagent, Taq DNA polymerase, Opti-MEM I medium, fetal calf serum, and normal horse serum were from Life Technologies, Inc. A Wizard genomic DNA purification kit was bought from Promega (Madison, WI). ABgene Total RNA isolation reagent was from Advanced Biotechnologies Ltd. (Surrey, UK). A QIAquick gel extraction kit was from Qiagen Pty. Ltd. (Clifton Hill, Victoria, Australia). A QuikChangeTM site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). Two types of mouse anti-human P2X7 receptor monoclonal antibodies (mAbs, clones L4 and B2) were used in this study. L4 was prepared from hybridoma supernatant by chromatography on protein A-Sepharose Fast Flow as described previously (14Buell G. Chessell I.P. Michel A.D. Collo G. Salazzo M. Herren S. Gretener D. Grahames C. Kaur R. Kosco-Vilbois M.H. Humphrey P.P. Blood. 1998; 92: 3521-3528Crossref PubMed Google Scholar). Purified B2 was kindly provided by Dr. Gary Buell. FITC labeling kits were used to conjugate these two P2X7 antibodies according to the manufacturer's instructions. The conjugated L4 had 1.2 FITC per IgG, and B2 had 1.1 FITC per IgG. Both anti-P2X7 antibodies showed no binding to the surface or cytoplasm of HEK293 cells, a cell line that does not express this receptor in subconfluent conditions (15Chessell I.P. Michel A.D. Humphrey P.P. Br. J. Pharmacol. 1998; 124: 1314-1320Crossref PubMed Scopus (113) Google Scholar). These two mAbs showed similar surface binding to human hemopoietic cells at a saturating concentration of 60 μg/ml. However, L4 strongly blocks P2X7 receptor function (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar, 14Buell G. Chessell I.P. Michel A.D. Collo G. Salazzo M. Herren S. Gretener D. Grahames C. Kaur R. Kosco-Vilbois M.H. Humphrey P.P. Blood. 1998; 92: 3521-3528Crossref PubMed Google Scholar), whereas B2 inhibits <7% of P2X7 receptor function. 2G. Buell, unpublished observation. Peripheral blood lymphocytes and monocytes were obtained from 45 normal subjects, and the patient with B-cell chronic lymphocytic leukemia has been reported in our previous study (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar). Mononuclear cells were separated on Ficoll-Hypaque, washed once, and resuspended in Hepes-buffered NaCl medium (145 mm NaCl, 5 mm KCl, 10 mm Hepes, pH 7.5, 5 mmd-glucose, 1 g/liter bovine serum albumin). In experiments on monocyte-derived macrophages, a mononuclear preparation was incubated for 12 h in plastic flasks and gently washed to remove nonadherent cells, and the adherent monocytes were cultured for 7 days in RPMI 1640 medium plus 10% human AB serum and 100 ng/ml interferon-γ. Macrophages were then detached by mechanical scraping for flow cytometric analysis. HEK-293 cells were cultured in RPMI 1640 complete medium supplemented with 10% heat-inactivated fetal calf serum, 2 mm glutamine, and 0.02 mg/ml gentamicin. Cells (2 × 106) prelabeled with appropriate fluorophore-conjugated anti-CD mAbs or anti-P2X7 mAb (clone B2) were washed once and resuspended in 1.0 ml of HEPES-buffered KCl medium (10 mm HEPES, 150 mm KCl, 5 mmd-glucose, 0.1% bovine serum albumin, pH 7.5) at 37 °C. Cells were analyzed at 1000 events per second on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and were gated by forward and side scatter and by cell type-specific antibodies. All samples were stirred, and the temperature was controlled at 37 °C using a Time Zero module (Cytek, Fremont, CA). Ethidium (25 μm) was added, followed 40 s later by addition of 1.0 mm ATP. The linear mean channel of fluorescence intensity for each gated subpopulation over successive 5-s intervals was analyzed by WinMDI software (Joseph Trotter, version 2.7) and plotted against time. To correct for any slight variation in the performance of the flow cytometer, fluorescent standard beads (Flow Cytometry Standards Corp., Research Triangle Park, NC) were analyzed each day (9Wiley J.S. Gargett C.E. Zhang W. Snook M.B. Jamieson G.P. Am. J. Physiol. 1998; 275: C1224-C1231Crossref PubMed Google Scholar). Lymphocytes (1 × 107/ml) were washed once and loaded with 2 μm Fura-2-acetoxymethyl ester by incubation at 37 °C for 30 min in Ca2+-free Hepes-buffered NaCl medium. Cells were washed once and incubated in Hepes-buffered NaCl with 1 mm Ca2+ for another 30 min. Lymphocytes were then washed twice and resuspended in 3 ml of Hepes-buffered KCl medium at a concentration of 2 × 106 cells/ml. These samples were stirred at 37 °C and stimulated with 1 mm ATP after addition of 1.0 mm BaCl2. Entry of Ba2+ into cells loaded with Fura-2 produces changes almost identical to those produced by Ca2+ in the excitation and emission spectra of Fura-2. Fluorescent signals were recorded on a Johnson Foundation fluorometer with excitation at 340 nm and emission at 500 nm. Calibration ofFmax and Fmin was performed after each run by adding 25 μm digitonin followed by 50 mm EGTA. Control experiments showed that addition of ATP did not release Ca2+ from the internal stores of lymphocytes suspended in medium containing EGTA. Genomic DNA was extracted from peripheral blood using the Wizard genomic DNA purification system. A primer pair was designed within exon 13 of the P2X7 gene to amplify a 356-base pair product from genomic DNA. P2X7oligonucleotides were synthesized by Life Technologies, Inc.. The forward primer was 5′-ACTCCTAGATCCAGGGATAGCC-3′, and the reverse primer was 5′-TCACTCTTCGGAAACTCTTTCC-3′. PCR amplification (35 cycles of denaturation at 95 °C for 45 s, annealing at 52 °C for 45 s, and extension at 72 °C for 1 min) produced a fragment of the expected 356-base pair size. PCR products were separated in 2% agarose gel and visualized by ethidium bromide staining. Amplified PCR products were purified using the QIAquick gel extraction kit protocol. Using an AmpliTaq FS dye terminator cycle sequencing kit (PerkinElmer Life Sciences), a fluorescence-based cycle sequencing reaction was performed to sequence the PCR products of P2X7 directly from both ends using specific primers. Sequencing electrophoresis was carried out on the ABI PRISM 377 DNA sequencer, and the ABI PRISM sequencing analysis software (version 3.0) was used for the analysis. The full-length clone of hP2X7 (GenBankTM accession number Y09561) was used in these studies. hP2X7 cDNA was kindly provided by Dr. Gary Buell as a NotI-NotI insert in pcDNA3 (Invitrogen). hP2X7 was removed from pcDNA3 using a NotI-NotI digest and ligated into pCI (Promega), which is a cytomegalovirus driven mammalian expression vector. Mutant 1513A→C (E496A) was introduced using overlap PCR (Quick ChangeTM site-directed mutagenesis kit, Stratagene) using the expression vector pCI-hP2X7 as a template. The P2X7 point mutation was constructed using a pair of complementary mutagenic primers, consisting of the mutagenic codon flanked by sequences homologous to the wild-type strand of the template. After digestion of the parental DNA with DpnI, intact mutation-containing synthesized DNA was transformed into competent DH5α cells. All mutations were confirmed by sequencing. The primer sequences were as follows: 1513A→C (E496A) forward, GG.TGC.CTG.GAG.G C G.CTG.TGC.TGC.CGG; 1513A→C (E496A) reverse, CCG.GCA.GCA.CAG.C G C.CTC.CAG.GCA.CC. Base changes introducing the mutations are in bold type and underlined. Full-length P2X7 cDNA in a plasmid vector pcDNA3 or the mutated P2X7 in pCI as above was transfected into HEK-293 cells by the LipofectAMINE 2000 Reagent. Transfection experiments always employed the minimum amount of cDNA that gave surface P2X7 expression. After 40–44 h, cells were collected by mechanical scraping in RPMI 1640 medium containing 10% fetal calf serum. Plastic nonadherent mononuclear cells were incubated on poly-l-lysine-coated (0.1 mg/ml) glass coverslips for 60 min. Fixed cells (4% paraformaldehyde) were blocked with 20% horse serum and 0.1% bovine serum albumin before incubating with anti-human P2X7 receptor mAb or isotype control antibody and subsequent labeling with Cy2-conjugated donkey anti-murine IgG antibody. Cells were visualized with a Leica TCS NT UV laser confocal microscope system as previously described (16Li G.H. Lee E.M. Blair D. Holding C. Poronnik P. Cook D.I. Barden J.A. Bennett M.R. J. Biol. Chem. 2000; 275: 29107-29112Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Lymphocytes (1 × 107/ml) were incubated with 200 μm BzATP for 15 min at 37 °C in Hepes-buffered NaCl medium, washed once, and incubated in RPMI 1640 medium with 10% fetal calf serum for 24 h. Cells were washed once and stained with FITC-anti-CD3 mAb and 7-amino-actinomycin D (20 μg/ml) for 20 min at room temperature. Viable and nonviable cells were measured by flow cytometry as previously described (17Philpott N.J. Turner A.J. Scopes J. Westby M. Marsh J.C. Gordon-Smith E.C. Dalgleish A.G. Gibson F.M. Blood. 1996; 87: 2244-2251Crossref PubMed Google Scholar). Our previous data have shown that P2X7 receptor function in monocyte or lymphocyte subsets can be measured by the ATP-induced uptake of ethidium at 37 °C using time-resolved two-color flow cytometry (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar). Mononuclear preparations from 32 normal subjects were pre-incubated with appropriate FITC-labeled monoclonal antibodies, and ATP-induced uptake of ethidium into gated monocyte and lymphocyte subpopulations was measured. Ethidium uptake through the P2X7 channel/pore was 5-fold greater for monocytes than for B-, T-, or NK-lymphocytes of normal origin, but for all cell types there was variation in the functional response of the P2X7receptor (Fig. 1). One subject showed a complete lack of P2X7 function in both monocytes and lymphocytes (shown in Fig. 1 by the filled circles). Variability in ATP-induced dye uptake into monocytes has been observed by others (13Lammas 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 (351) Google Scholar). Because the long C-terminal tail of the P2X7 receptor regulates its permeability properties, the sequence of genomic DNA corresponding to this region was analyzed. Thus a PCR product was amplified directly from DNA between nucleotides 1425 and 1780 of the coding region of the P2X7 gene, and the product was sequenced. In 9 of 45 subjects a heterozygous nucleotide substitution (adenine to cytosine) was found at position 1513, whereas in 1 of 45 subjects a homozygous 1513A→C substitution was observed (Fig.2). Because the fractional frequency of the mutant allele was 11 of 90 (0.122) in the Caucasian population, it fulfils the criterion for a single nucleotide polymorphism. The deduced amino acid change for this mutation is glutamic acid to alanine at amino acid 496 (E496A) of the P2X7 protein. Skin fibroblasts were cultured from a punch biopsy of skin from the homozygous normal subject, DNA was extracted, and a product was amplified using primers for the C-terminal tail of the P2X7gene. Sequence analysis of the product showed only cytosine to be present at position 1513 (results not shown). Large amounts of P2X7 protein are found in an intracellular location in monocytes and lymphocytes of all subtypes (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar), and we studied whether the 1513A→C mutation may have reduced the surface expression of this receptor. Confocal microscopy showed strong surface expression of the P2X7 receptor on lymphocytes from subjects who were germline or homozygous for this mutation (Fig. 3), and monocytes showed a similar strong surface P2X7 expression (data not shown). Flow cytometric measurement of P2X7 expression using a monoclonal antibody to the extracellular domain of P2X7(14Buell G. Chessell I.P. Michel A.D. Collo G. Salazzo M. Herren S. Gretener D. Grahames C. Kaur R. Kosco-Vilbois M.H. Humphrey P.P. Blood. 1998; 92: 3521-3528Crossref PubMed Google Scholar) showed that the surface expression of this receptor on either B- or T-lymphocytes from heterozygous or homozygous patients was not significantly different from B- or T-lymphocytes that were of germline sequence at position 1513 (Table I).Table IExpression and function of P2X7 receptor in mononuclear cells from 20 normal subjects and 1 B-CLL subject with germline and 1513A→C mutant P2X7Status at 1513nRelative P2X7 expression (mean channels of fluorescence intensity ± S.D.)P2X7 function (arbitrary units of area under ATP-induced ethidium uptake curve at 5 min ± S.D.)B-lymT-lymB-lymT-lymNK cellsMonocytesNormal subjectsGermline (A/A)129.3 ± 2.77.1 ± 3.45393 ± 20013871 ± 20835226 ± 189028076 ± 5357Heterozygotes (A/C)79.8 ± 2.08.7 ± 7.12121 ± 1441 1-aP value (versusGermline) is 0.0011, 0.0045, 0.025, and 0.00026, respectively.1341 ± 690 1-aP value (versusGermline) is 0.0011, 0.0045, 0.025, and 0.00026, respectively.3062 ± 1999 1-aP value (versusGermline) is 0.0011, 0.0045, 0.025, and 0.00026, respectively.13900 ± 7162 1-aP value (versusGermline) is 0.0011, 0.0045, 0.025, and 0.00026, respectively.Homozygote (C/C)112.46.0266101320777B-CLL subjectHomozygote (C/C)19.86.53084037155Mononuclear cell preparations (1 × 107/ml) from normal or B-CLL subjects were incubated for 20 min at 20 °C with fluorescein-conjugated P2X7 mAb (clone L4) plus RPE-labeled CD3 and RPE-Cy5-labeled CD19 surface marker antibody in Hepes-buffered saline containing 10% group AB serum. lym, lymphocytes.1-a P value (versusGermline) is 0.0011, 0.0045, 0.025, and 0.00026, respectively. Open table in a new tab Mononuclear cell preparations (1 × 107/ml) from normal or B-CLL subjects were incubated for 20 min at 20 °C with fluorescein-conjugated P2X7 mAb (clone L4) plus RPE-labeled CD3 and RPE-Cy5-labeled CD19 surface marker antibody in Hepes-buffered saline containing 10% group AB serum. lym, lymphocytes. The function of P2X7 receptors expressed on lymphocytes or monocytes was compared with the genotype at position 1513 of the P2X7 gene. Typical ethidium uptake curves for monocytes and B-, T-, and NK-lymphocytes are shown in Fig.4 for normal subjects with germline, heterozygous, or homozygous DNA at position 1513. A single patient from our previous study (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar) with B-cell chronic lymphocytic leukemia and homozygous 1513A→C is included in Fig. 4 and Table I for comparison. Homozygosity for the mutation led to an almost complete loss of function of the receptor, whereas heterozygosity for the mutation gave a function approximately half that of the germline P2X7 sequence (Fig. 4, a–d). Measurement of P2X7 function in a larger group of subjects (n = 20, Table I) showed that the mean ATP-induced ethidium uptake was reduced in heterozygous subjects to half the uptake found in subjects with germline sequence, and this magnitude of reduction was found for the four cell types studied (monocytes,p < 0.001; B-, p < 0.002; T-,p < 0.005; and NK-lymphocytes, p < 0.03). We also studied ATP-induced uptake of Ba2+into lymphocytes prepared from the subject with homozygous mutant P2X7. These cells failed to respond to ATP (Fig.5), indicating that the mutant P2X7 channel was nonfunctional to small inorganic cations as well as ethidium+ as permeants (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197Crossref PubMed Google Scholar).Figure 5Ba2+ influx on lymphocytes from subjects with germline and 1513A→C homozygous and heterozygous mutant P2X7. Lymphocytes (6 × 106) loaded with 2 μm Fura-2-acetoxymethyl ester were resuspended in 3 ml of Hepes-buffered KCl medium. 1.0 mm Ba2+was added 40 s before stimulation with 1.0 mm ATP as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) cDNA for germline P2X7 or P2X7carrying the 1513A→C mutation was transfected into HEK293 cells to study whether this mutation abolishes function in transfection experiments. At 40 h after transfection the surface expression of the P2X7 receptor was quantitated by the binding of FITC-conjugated mAb (clone B2), and the ATP-induced uptake of ethidium was studied in the same cell population by two-color flow cytometry. Preliminary experiments suggested that the function of the mutated P2X7 depended on the density at which this receptor was expressed on the cell surface. For this reason a gating strategy was adopted in which cells expressing a zero, low, or high density of P2X7 receptors were analyzed as three separate populations (Fig. 6, a–c). The cohort of cells with negative P2X7 expression showed no ATP-induced ethidium uptake with either germline or mutated cDNA (Fig.6 d). The cohort of cells with low expression of the P2X7 receptor showed strong ATP-induced ethidium uptake in the germline P2X7, but the mutant P2X7 had no function (Fig. 6 e). However, in the cohort of cells with the highest P2X7 surface expression, substantial ATP-induced ethidium uptake was observed for both the germline and, to a lesser extent, the mutant P2X7 (Fig. 6 f). These data suggest that the impaired function of the P2X7 receptor in cells carrying the E496A mutation could be reversed when the density of the mutant receptor was increased on the cell surface. Differentiation of monocytes into macrophages greatly increases both the expression and function of the P2X7receptor (18Humphreys B.D. Dubyak G.R. J. Leukocyte Biol. 1998; 64: 265-273Crossref PubMed Scopus (127) Google Scholar, 19Hickman S.E. el Khoury J. Greenberg S. Schieren I. Silverstein S.C. Blood. 1994; 84: 2452-2456Crossref PubMed Google Scholar). Peripheral blood monocytes were cultured with interferon-γ for 7 days, and the function of P2X7receptor was measured in the CD14+ macrophage population. Macrophages from subjects with germline P2X7 showed an ATP-induced ethidium uptake about 5-fold greater than their precursor monocytes (Fig. 7 a). Thus the area under the ATP-induced ethidium uptake curve increased from 28,920 units on day 0 to 160,000 units in day 6 macrophages. Macrophages from a subject homozygous for the 1513A→C polymorphism developed partial P2X7 function compared with the absent function in the precursor monocytes (Fig. 7 b; 0 units on day 0 to 30,800 units on day 6). Although the P2X7 expression (mean channel fluorescence intensity) on germline monocytes (48) increased after maturation to macrophages (284), this increase was less in the homozygous mutant cells (from 51 to 96). Thus the functional defect associated with the E496A polymorphism in cells of monocytic lineage could be partially reversed when the abundance of native P2X7 was increased on the cell surface. P2X7-mediated cytotoxicity was studied in lymphocytes from subjects who were germline or homozygous for the E496A polymorphism. A mononuclear preparation of peripheral blood was exposed to BzATP for 15 min, washed, and incubated a further 24 h prior to assay by two-color flow cytometry using (a) 7-amino-actinomycin D as a viability dye and (b) FITC-conjugated CD3 mAb to gate on the predominant T-lymphocyte subpopulation. The fluorescent dot-plots (Fig.8) identify two distinct populations of viable cells (lower region) and nonviable cells (upper region) after 24 h of incubation. The percentage of nonviable cells was markedly reduced in the homozygote P2X7 mutant compared with germline T-cells (Fig. 8, a and b). In control lymphocytes not exposed to BzATP, the percentage of nonviable cells was 3.2 for germline and 6.6 for homozygote after 24 h of incubation. The data in this study show that the function of the human P2X7 receptor is affected by a single nucleotide mutation of adenine to cytosine at position 1513 of cDNA, which changes glutamic acid to alanine at amino acid position 496. Homozygosity (C/C) for this polymorphic mutation led to almost complete loss of P2X7 function in leukocytes, whereas heterozygosity (A/C) gave a function that was half that of cells with the germline P2X7 sequence. The negative effect of this mutation on P2X7 function was evident in all leukocytes that express surface P2X7, namely monocytes, B-lymphocytes, T-lymphocytes, and NK-cells. Polymorphonuclear leukocytes and platelets that have only weak or no surface expression of P2X7 (12Gu B.J. Zhang W.Y. Bendall L.J. Chessell I.P. Buell G.N. Wiley J.S. Am. J. Physiol. 2000; 279: C1189-C1197