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

Ben J. GuDagger , Weiyi ZhangDagger , Rebecca A. Worthington§, Ronald SluyterDagger , Phuong Dao-UngDagger , Steven Petrou, Julian A. Barden§, and James S. WileyDagger ||

From the Departments of Dagger  Medicine and § Anatomy and Histology, University of Sydney, Sydney, New South Wales 2006 and the  Department of Physiology, University of Melbourne, Parkville, Victoria 3050, Australia

Received for publication, November 15, 2000, and in revised form, January 8, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1513Aright-arrowC) 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-gamma , which up-regulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1), macrophages (2), and lymphocytes (3, 4). The P2X7 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 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 (7). 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 (8). 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 (9) and the activation of various proteases including membrane metalloproteases (10) and intracellular caspases (2, 11). 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 (9). In normal leukocytes a close correlation has been found between ATP-induced ethidium uptake and the surface expression of P2X7 receptors measured by the binding of a fluorescein isothiocyanate (FITC)1-conjugated antibody to the extracellular domain of this receptor (12).

There is increasing evidence that a genetic factor plays a role in the functional phenotype of the P2X7 receptor. Thus, Lammas et al. (13) 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 (12). 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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 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).

Antibody Production and Preparation-- 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 (14). 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 (15). 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 (12, 14), whereas B2 inhibits <7% of P2X7 receptor function.2

Source of Human Leukocytes-- 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 (12). 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 mM D-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-gamma . Macrophages were then detached by mechanical scraping for flow cytometric analysis.

Cultured HEK-293 Cells-- 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.

Ethidium Influx Measurement by Flow Cytometry-- 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 mM D-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 (9).

Cytosolic Ba2+ Measurements by Fluorometry-- 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 of Fmax 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.

DNA Extraction and PCR-- 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. P2X7 oligonucleotides 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.

DNA Sequencing for PCR Products-- 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.

Site-directed Mutagenesis-- 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 1513Aright-arrowC (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 DH5alpha cells. All mutations were confirmed by sequencing. The primer sequences were as follows: 1513Aright-arrowC (E496A) forward, GG.TGC.CTG.GAG.GCG.CTG.TGC.TGC.CGG; 1513Aright-arrowC (E496A) reverse, CCG.GCA.GCA.CAG.CGC.CTC.CAG.GCA.CC. Base changes introducing the mutations are in bold type and underlined.

P2X7 Transfection into HEK293 Cells-- 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.

Immunofluorescent Staining and Confocal Microscopy-- 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 (16).

ATP-induced Cytotoxicity Assay-- 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 (17).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

P2X7 Function in Monocytes and Lymphocytes-- 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 (12). 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 P2X7 receptor (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 (13).



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Fig. 1.   Variability of P2X7 receptor function in normal human peripheral blood mononuclear cell subsets. Mononuclear preparations (2 × 106 cells/ml) prelabeled with cell type-specific antibodies (CD14 for monocytes, CD19 for B-lymphocytes, CD3 for T-lymphocytes, and CD16 for NK cells) were suspended in Hepes-buffered KCl medium at 37 °C. Ethidium (25 µM) was added, followed 40 s later by 1.0 mM ATP. The mean channel of cell-associated fluorescence intensity was measured for each gated population at 5-s intervals. P2X7 function is shown as arbitrary units of area under the ATP-induced ethidium uptake curve in the first 5 min of incubation. lym, lymphocytes.

Identification of a Single Nucleotide Polymorphism in the C-terminal Tail of the P2X7 Gene-- 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 1513Aright-arrowC 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.



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Fig. 2.   Sequence of genomic DNA at the C-terminal tail of the P2X7 gene showing (a) germline, (b) 1513Aright-arrowC homozygous mutant, and (c) 1513Aright-arrowC heterozygote. A 356-base pair product was amplified by using a primer pair within exon 13 of the P2X7 gene (forward, 5'-ACTCCTAGATCCAGGGATAGCC-3'; reverse, 5'-TCACTCTTCGGAAACTCTTTCC-3'). PCR products were purified and sequenced directly from both ends using the same primers.

The 1513Aright-arrowC Mutation Is Present in Skin Fibroblasts-- 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 P2X7 gene. Sequence analysis of the product showed only cytosine to be present at position 1513 (results not shown).

Surface Expression of P2X7 Is Not Affected by the Polymorphism-- Large amounts of P2X7 protein are found in an intracellular location in monocytes and lymphocytes of all subtypes (12), and we studied whether the 1513Aright-arrowC 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 (14) 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).



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Fig. 3.   Confocal images of P2X7 receptor expression on the surface of lymphocytes. Lymphocytes from normal subjects, either (a) germline or (b) 1513Aright-arrowC homozygote, were labeled with anti-P2X7 receptor mAb (clone L4) and subsequently with Cy2-conjugated anti-mouse IgG antibody. Isotype control antibody was included as a negative control and showed no staining. The calibration bar is 2 µm.


                              
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Table I
Expression and function of P2X7 receptor in mononuclear cells from 20 normal subjects and 1 B-CLL subject with germline and 1513Aright-arrowC mutant P2X7
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.

Correlation of P2X7 Function with the Polymorphism-- 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 (12) with B-cell chronic lymphocytic leukemia and homozygous 1513Aright-arrowC 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 (12).



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Fig. 4.   Typical ATP-induced ethidium uptake curve in mononuclear cell subsets from normal subjects with germline, 1513Aright-arrowC heterozygous, and 1513Aright-arrowC homozygous P2X7 as indicated. A single patient with B-chronic lymphocytic leukemia who was 1513Aright-arrowC homozygous P2X7 is included for comparison. 2 × 106 cells prelabeled with appropriate FITC-conjugated surface marker antibody were incubated in 1 ml of Hepes-buffered KCl at 37 °C. Ethidium bromide (25 µM) was added, followed 40 s later by 1 mM ATP. The mean channel of cell-associated fluorescence intensity was measured at 5-s intervals for (a) monocyte (gated CD14+), (b) B-lymphocyte (gated CD19+), (c) T-lymphocyte (gated CD3+), and (d) NK cell (gated CD16+) populations.



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Fig. 5.   Ba2+ influx on lymphocytes from subjects with germline and 1513Aright-arrowC 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.

Function of 1513Aright-arrowC Mutated P2X7 Transfected into HEK293-- cDNA for germline P2X7 or P2X7 carrying the 1513Aright-arrowC 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. 6d). 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. 6e). 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. 6f). 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.



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Fig. 6.   Surface expression (a-c) and function (d-f) of HEK293 cells transiently transfected with germline and 1513Aright-arrowC site-directed mutant human P2X7 cDNA. 1 µg of DNA and 6 µl of LipofectAMINE 2000 reagent were incubated in 300 µl of Opti-MEM I medium for 20 min. The mixture was then added to a 25-cm2 flask containing 5 × 106 HEK293 cells in 3 ml of medium. Cells were collected after 40-44 h, washed once, and incubated with 10 µg/ml FITC-conjugated mouse anti-human P2X7 mAb (clone B2) for 15 min at room temperature. Cells were then washed once and resuspended in 1 ml of Hepes-buffered KCl medium. Ethidium bromide (25 µM) was added, followed 40 s later by 1 mM ATP. The mean channel of cell-associated fluorescence intensity was measured at 5-s intervals for gated subpopulations, which expressed P2X7 receptors on their surface at (d) zero, (e) low, and (f) high density levels. Data were collected at a constant flow rate of 1,000 total events per second. The gating windows R1, R2, and R3 were exactly the same for germline and mutant cells. Control experiments showed negative expression and function of P2X7 on native HEK293 cells. Control experiments excluded an inhibitory effect of B2 monoclonal antibody on P2X7 function. a-c, the dotted lines are the isotype controls, and the solid lines show the histogram for FITC-P2X7 mAb.

Homozygous Mutant P2X7 Regains Partial Function in Macrophages-- Differentiation of monocytes into macrophages greatly increases both the expression and function of the P2X7 receptor (18, 19). Peripheral blood monocytes were cultured with interferon-gamma for 7 days, and the function of P2X7 receptor 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. 7a). 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 1513Aright-arrowC polymorphism developed partial P2X7 function compared with the absent function in the precursor monocytes (Fig. 7b; 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.



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Fig. 7.   ATP-induced ethidium uptake on fresh monocytes and monocyte-derived macrophages from normal subjects with germline and 1513Aright-arrowC homozygous P2X7. Monocytes from mononuclear preparations were allowed to adhere to plastic culture flasks overnight and were cultured for another 6 days in medium plus 100 ng/ml interferon-gamma . Cells were collected by gentle mechanical scraping and labeled with FITC-anti-CD14 mAb. The linear mean channel fluorescence intensity was measured in each 5-s interval on the gated CD14+ population after 25 µM ethidium and 1 mM ATP were added.

ATP-induced Cytotoxicity Is Impaired by the Homozygous P2X7 Polymorphism-- 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.



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Fig. 8.   BzATP-induced cytotoxicity of T-lymphocytes from subjects with germline and 1513Aright-arrowC homozygous P2X7. Lymphocytes (1 × 107/ml) were incubated with or without 200 µM BzATP in Hepes buffer, NaCl medium at 37 °C for 15 min, washed once, and resuspended in RPMI 1640 medium with 10% fetal calf serum. After 24 h of incubation cells were labeled with FITC-anti-CD3 mAb and the viability dye 7-amino-actinomycin D (7-AAD). The percentage of live cells is shown in the lower region of each dot-plot, and the percentage of nonviable cells is shown in the upper region of each dot-plot.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (12) were not tested. The finding that skin fibroblasts from a homozygous 1513Aright-arrowC subject also carry only cytosine at position 1513 indicates the constitutional nature of this mutation, which affects tissues other than leukocytes. The fractional frequency of the mutant 1513Aright-arrowC allele in the normal subjects was 0.122, so that the mutant allele falls within the definition of a single nucleotide polymorphism defined by a prevalence greater than 0.01 (1%) in the population. Application of the Hardy-Weinberg equilibrium to the 1513Aright-arrowC allele frequency found at the DNA level yields an expected value of 0.7 homozygotes, which is not significantly different from the observed value of 1 (X2 = 0.21 with one degree of freedom). Single nucleotide polymorphisms are increasingly recognized as a source of genetic variation, and their density may be as high as one per kilobase of cDNA (20). Most of these polymorphisms have a neutral effect on function, but some contribute to loss of protein function, such as was found with this 1513Aright-arrowC allele. In a recent study of B-cell chronic lymphocytic leukemia, our group found that 3 of 12 patients demonstrated low or absent function of the P2X7 receptor in the malignant B-lymphocytes as well as in the normal monocytes of peripheral blood (12). One of these three patients carried the 1513Aright-arrowC mutant P2X7 allele in homozygous dosage, whereas the other two patients were germline at this position.3 Clearly 1513Aright-arrowC is only one of several genetic changes that can inhibit the function of the P2X7 receptor. It has been previously reported that truncation of the long C-terminal tail of the rat P2X7 receptor abolishes ATP-induced uptake of large fluorescent dyes such as Yo-Pro2+ (8), and truncation of the C-terminal tail of the human P2X7 receptor also abolishes ATP-induced channel/pore formation.4 For this reason we sought a loss-of-function mutation in the carboxyl terminus of P2X7. Truncation of a receptor often leads to failure of its surface expression, such as that shown for the 32-base pair deletion in the chemokine receptor CCR5 gene (21) or for the 49-amino acid deletion from the carboxyl terminus of the sulfonylurea receptor, which prevents trafficking of this receptor and its associated ATP-sensitive K+ channel to the surface of the pancreatic beta -cell (22). However, the E496A polymorphism in P2X7 allows full expression of the mutant receptor on the cell surface, as shown in Fig. 3 and Table I for both B- and T-lymphocytes.

The polymorphic 1513Aright-arrowC mutation of P2X7 changes glutamic acid to alanine at amino acid 496 (E496A), and the present data suggest that this glutamic acid residue at position 496 is centrally involved in the interactions that lead to formation of the P2X7 channel/pore. The molecular mechanisms underlying the opening of the cation channel and its transition to a fully dilated pore are not resolved. The simplest model for pore dilation is that it is an intrinsic property of the P2X7 receptor that involves a small scale structural change, perhaps in the selectivity filter of the channel (23). Alternative views suggest that pore dilation involves a large scale structural change, such as that induced by the dynamic addition of subunits to the existing oligomeric structure (24) or by an interaction with a protein partner, or the activation of a molecularly distinct pore protein (25) by ligated P2X7 receptor. The inability of some oocyte expression systems to display BzATP-activated pore formation (26, 27) also provides evidence for regulation of the pore dilation. The finding that the homozygous mutant P2X7 receptor is nonfunctional for both a small cation, permeant Ba2+, as well as the larger ethidium+ emphasizes the importance of this glutamic acid at position 496, both for immediate channel opening and its dilatation to a pore. The simplest explanation of the present data is that the glutamic to alanine substitution in the mutant P2X7 weakens the electrostatic interactions governing the assembly of the P2X7 channel complex in the plasma membrane.

Transfection of 1513Aright-arrowC mutant P2X7 into HEK293 demonstrated that the loss of channel function in mutant P2X7 could be reversed at high levels of surface expression of the mutant receptor. Two-color flow cytometry (Fig. 6) was used to directly compare ethidium influx through the P2X7 pore in transfected cells gated into three subpopulations: those expressing no receptor and those expressing low and high levels of this receptor. This gating strategy employed an FITC-conjugated mAb (clone B2) that binds to an extracellular epitope of P2X7 but does not inhibit the function of the receptor. Thus ATP-induced ethidium uptake was measured on the red (570 nm) channel into two cell populations defined by high and low (R3 and R2, respectively) fluorescence on the green FITC (525 nm) channel. High fluorescence (R3) corresponds to high surface expression of P2X7, whereas low fluorescence (R2) indicates low surface expression of P2X7. Germline P2X7 showed function at both high and low receptor numbers at the plasma membrane. In contrast, the mutant P2X7 was nonfunctional at low numbers but regained partial function at a higher density of expressed receptors. This important finding was confirmed for the native P2X7 receptor, which is up-regulated when monocytes from peripheral blood are cultured with interferon-gamma to produce macrophages (Fig. 7). The function of germline P2X7 was stimulated about 5-fold in macrophages compared with their precursor monocytes, but the mutant P2X7 only regained partial function in macrophages compared with its zero function in precursor monocytes. Increased receptor abundance may explain the partial restoration of mutant P2X7 channel function, because raising the receptor numbers in the membrane of either HEK293 cells (Fig. 6) or human macrophages (Fig. 7) would tend to compensate for weakened self-associations and promote receptor assembly by a direct mass action effect. Whatever the mechanism of P2X7 assembly in the membrane, the data in Table I shows that much of the person-to-person variation in P2X7 function can be explained by the genetic polymorphism at amino acid position 496 of the P2X7 receptor molecule.

Both gain-of-function as well as loss-of-function mutations can affect genes encoding ion channel proteins (23). Thus an asparagine to lysine polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor enhances coupling to Gi in the presence of agonist (28). Three loss-of-function mutations have been identified in the human K1R6.2 gene, which encodes the two-transmembrane protein subunit of the pancreatic beta -cell ATP-sensitive K+ channel (22). Loss-of-function mutations occur in the nompC gene, a six-transmembrane ion channel in Drosophila responsible for mechanosensory signaling (29). However, few if any genetic polymorphisms have been described previously in which one allele encodes a nonfunctional channel.

Extracellular ATP has an emerging role in the immune system, because P2X7 activation leads to apoptotic death of thymocytes (30, 31), B-lymphocytes (4), macrophages (2), and dendritic cells (32, 33). Thus incubation of mononuclear cells from peripheral blood with ATP gave substantial apoptotic death of T-lymphocytes, but cell death was greatly attenuated in T-lymphocytes from the subject with homozygous E496A P2X7 protein (Fig. 8). There is good evidence that activation of macrophage P2X7 receptors by ATP can produce killing of intracellular Mycobacteria tuberculosis by these cells (13, 34). Stimulation of phospholipase D appears to be involved in the killing mechanism (35), and one of the consequences of P2X7 activation is stimulation of the activity of phosphatidyl choline-specific phospholipase D (36-40). Other downstream effects of the P2X7 receptor activation may also occur, such as the generation of reactive oxygen intermediates and the stimulation of intracellular caspases that not only kill the organism but also lead to the apoptotic death or cytolysis of the host cell. It is possible that the polymorphism described above may be one of the susceptibility factors predisposing individuals to Mycobacteria infections. Thus study of the P2X7 knockout mouse (41) and its resistance to certain infections requiring competent macrophages for control will be important in defining a role for this receptor. Regardless of the clinical associations of the polymorphism at amino acid 496, this loss-of-function mutation affecting the C-terminal tail of P2X7 may help unravel the molecular events leading to channel/pore formation.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Gary Buell and Dr. Iain Chessell for gifts of monoclonal antibodies, Dr. Zhan-he Wu for fibroblast culture, and Shelley Spicer for typing the manuscript.


    FOOTNOTES

* This work was supported by the Sydney University Cancer Research Fund, the New South Wales Cancer Council, The Leo & Jenny Foundation, and the Cecilia Kilkeary Foundation Ltd.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Clinical Sciences Bldg., Nepean Hospital, Penrith, NSW 2750, Australia. Tel.: 61-2-473-43277; Fax: 61-2-473-43432; E-mail: wileyj@medicine.usyd.edu.au.

Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M010353200

2 G. Buell, unpublished observation.

3 B. J. Gu, W. Zhang, and J. S. Wiley, unpublished observation.

4 B. J. Gu and J. S. Wiley, unpublished observation.


    ABBREVIATIONS

The abbreviations used are: FITC, fluorescein isothiocyanate; Bz, benzoylbenzoyl; PCR, polymerase chain reaction; mAb, monoclonal antibody; HEK, human embryonic kidney; CD, cluster of differentiation.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Grahames, C. B., Michel, A. D., Chessell, I. P., and Humphrey, P. P. (1999) Br. J. Pharmacol. 127, 1915-1921[Abstract/Free Full Text]
2. Humphreys, B. D., Rice, J., Kertesy, S. B., and Dubyak, G. R. (2000) J. Biol. Chem. 275, 26792-26798[Abstract/Free Full Text]
3. Chused, T. M., Apasov, S., and Sitkovsky, M. (1996) J. Immunol. 157, 1371-1380[Abstract]
4. Peng, L., Bradley, C. J., and Wiley, J. S. (1998) Chin. Med. J. (Engl. Ed.) 78, 508-511
5. Nicke, A., Baumert, H. G., Rettinger, J., Eichele, A., Lambrecht, G., Mutschler, E., and Schmalzing, G. (1998) EMBO J. 17, 3016-3028[Abstract/Free Full Text]
6. Torres, G. E., Egan, T. M., and Voigt, M. M. (1999) J. Biol. Chem. 274, 6653-6659[Abstract/Free Full Text]
7. Rassendren, F., Buell, G. N., Virginio, C., Collo, G., North, R. A., and Surprenant, A. (1997) J. Biol. Chem. 272, 5482-5486[Abstract/Free Full Text]
8. Surprenant, A., Rassendren, F., Kawashima, E., North, R. A., and Buell, G. (1996) Science 272, 735-738[Abstract]
9. Wiley, J. S., Gargett, C. E., Zhang, W., Snook, M. B., and Jamieson, G. P. (1998) Am. J. Physiol. 275, C1224-C1231[Medline] [Order article via Infotrieve]
10. Gu, B., Bendall, L. J., and Wiley, J. S. (1998) Blood 92, 946-951[Abstract/Free Full Text]
11. Ferrari, D., Los, M., Bauer, M. K., Vandenabeele, P., Wesselborg, S., and Schulze-Osthoff, K. (1999) FEBS Lett. 447, 71-75[CrossRef][Medline] [Order article via Infotrieve]
12. Gu, B. J., Zhang, W. Y., Bendall, L. J., Chessell, I. P., Buell, G. N., and Wiley, J. S. (2000) Am. J. Physiol. 279, C1189-C1197
13. Lammas, D. A., Stober, C., Harvey, C. J., Kendrick, N., Panchalingam, S., and Kumararatne, D. S. (1997) Immunity 7, 433-444[Medline] [Order article via Infotrieve]
14. Buell, G., Chessell, I. P., Michel, A. D., Collo, G., Salazzo, M., Herren, S., Gretener, D., Grahames, C., Kaur, R., Kosco-Vilbois, M. H., and Humphrey, P. P. (1998) Blood 92, 3521-3528[Abstract/Free Full Text]
15. Chessell, I. P., Michel, A. D., and Humphrey, P. P. (1998) Br. J. Pharmacol. 124, 1314-1320[Abstract]
16. Li, G. H., Lee, E. M., Blair, D., Holding, C., Poronnik, P., Cook, D. I., Barden, J. A., and Bennett, M. R. (2000) J. Biol. Chem. 275, 29107-29112[Abstract/Free Full Text]
17. Philpott, N. J., Turner, A. J., Scopes, J., Westby, M., Marsh, J. C., Gordon-Smith, E. C., Dalgleish, A. G., and Gibson, F. M. (1996) Blood 87, 2244-2251[Abstract/Free Full Text]
18. Humphreys, B. D., and Dubyak, G. R. (1998) J. Leukocyte Biol. 64, 265-273[Abstract]
19. Hickman, S. E., el Khoury, J., Greenberg, S., Schieren, I., and Silverstein, S. C. (1994) Blood 84, 2452-2456[Abstract/Free Full Text]
20. Collins, A., Lonjou, C., and Morton, N. E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15173-15177[Abstract/Free Full Text]
21. Biti, R., Ffrench, R., Young, J., Bennetts, B., Stewart, G., and Liang, T. (1997) Nat. Med. 3, 252-253[Medline] [Order article via Infotrieve]
22. Sharma, N., Crane, A., Gonzalez, G., Bryan, J., and Aguilar-Bryan, L. (2000) Kidney Int. 57, 803-808[CrossRef][Medline] [Order article via Infotrieve]
23. Lester, H. A., and Karschin, A. (2000) Annu. Rev. Neurosci. 23, 89-125[CrossRef][Medline] [Order article via Infotrieve]
24. Tatham, P. E., and Lindau, M. (1990) J. Gen. Physiol. 95, 459-476[Abstract]
25. Nuttle, L. C., and Dubyak, G. R. (1994) J. Biol. Chem. 269, 13988-13996[Abstract/Free Full Text]
26. Klapperstuck, M., Buttner, C., Bohm, T., Schmalzing, G., and Markwardt, F. (2000) Biochim. Biophys. Acta 1467, 444-456[Medline] [Order article via Infotrieve]
27. Petrou, S., Ugur, M., Drummond, R. M., Singer, J. J., and Walsh, J. V., Jr. (1997) FEBS Lett. 411, 339-345[CrossRef][Medline] [Order article via Infotrieve]
28. Small, K. M., Forbes, S. L., Brown, K. M., and Liggett, S. B. (2000) J. Biol. Chem. 275, 38518-38523[Abstract/Free Full Text]
29. Walker, R. G., Willingham, A. T., and Zuker, C. S. (2000) Science 287, 2229-2234[Abstract/Free Full Text]
30. Zheng, L. M., Zychlinsky, A., Liu, C. C., Ojcius, D. M., and Young, J. D. (1991) J. Cell Biol. 112, 279-288[Abstract]
31. Nagy, P. V., Feher, T., Morga, S., and Matko, J. (2000) Immunol. Lett. 72, 23-30[CrossRef][Medline] [Order article via Infotrieve]
32. Nihei, O. K., de Carvalho, A. C., Savino, W., and Alves, L. A. (2000) Blood 96, 996-1005[Abstract/Free Full Text]
33. Coutinho-Silva, R., Persechini, P. M., Bisaggio, R. D., Perfettini, J. L., Neto, A. C., Kanellopoulos, J. M., Motta-Ly, I., Dautry-Varsat, A., and Ojcius, D. M. (1999) Am. J. Physiol. 276, C1139-C1147[Abstract/Free Full Text]
34. Sikora, A., Liu, J., Brosnan, C., Buell, G., Chessell, I., and Bloom, B. R. (1999) J. Immunol. 163, 558-561[Abstract/Free Full Text]
35. Kusner, D. J., and Adams, J. (2000) J. Immunol. 164, 379-388[Abstract/Free Full Text]
36. Kusner, D. J., and Dubyak, G. R. (1994) Biochem. J. 304, 485-491[Medline] [Order article via Infotrieve]
37. Humphreys, B. D., and Dubyak, G. R. (1996) J. Immunol. 157, 5627-5637[Abstract]
38. el-Moatassim, C., and Dubyak, G. R. (1992) J. Biol. Chem. 267, 23664-23673[Abstract/Free Full Text]
39. Gargett, C. E., Cornish, E. J., and Wiley, J. S. (1996) Biochem. J. 313, 529-535[Medline] [Order article via Infotrieve]
40. Fernando, K. C., Gargett, C. E., and Wiley, J. S. (1999) Arch. Biochem. Biophys. 362, 197-202[CrossRef][Medline] [Order article via Infotrieve]
41. Solle, M., Labasi, J., Perregaux, D. G., Stam, E., Petrushova, N., Koller, B. H., Griffiths, R. J., and Gabel, C. A. (2001) J. Biol. Chem. 276, 125-132[Abstract/Free Full Text]


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