From the Department of Medicine, University of Sydney
at Nepean Hospital, Penrith, New South Wales 2750, the
¶ Department of Physiology, University of Melbourne, Parkville,
Victoria 3050, and the
Department of Anatomy and Histology,
University of Sydney, Sydney, New South Wales 2006, Australia
Received for publication, December 16, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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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- The purinergic P2X7 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 Ca2+ and Na+ influx
and K+ 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). P2X7 activation also leads to extensive membrane blebbing (10), which is a typical morphological feature of the apoptotic process. P2X7
receptors have two transmembrane domains with intracellular amino and
carboxyl termini, and the P2X7 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
P2X7 receptor because truncation of this tail abolishes
ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12).
P2X7 has an oligomeric structure in the membrane based on
trimeric or larger complexes of identical subunits (13, 14), and there
is evidence that P2X7 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 P2X7
primary structure has been proposed as being important for maintaining
the full activity of the P2X7 channel (15).
A number of regulatory domains or motifs have been identified in the
intracellular carboxyl tail based on homology with other proteins.
These include a potential Src homology 3 binding domain (amino
acids 450-456) and an ankyrin repeat motif (amino acids 494-508)
(16). Ankyrin repeats have been shown to play a major structural role
in protein anchoring to the membrane or cytoskeleton, protein folding,
and protein-protein interaction (17, 18). In addition, a
lipopolysaccharide (LPS)1
binding motif with homology to the LPS-binding protein of plasma (16)
has been proposed between amino acids 573 and 590, whereas we have
shown that a region within this motif (amino acids 551-581) is
necessary for the surface expression of P2X7 (19). Upstream from this motif we have identified a single nucleotide polymorphism (1513A Materials--
ATP, ethidium bromide, BaCl2,
D-glucose, bovine serum albumin, RPMI 1640 medium,
gentamicin, collagen (Type X), and glycerol gelatin mounting medium
were purchased from Sigma. Interferon- Source of Human Leukocytes--
Peripheral blood mononuclear
cells were isolated by density gradient centrifugation as described
(22). Cells were resuspended in HEPES-buffered NaCl medium (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 5 mM D-glucose, and 1 mg/ml bovine serum albumin,
pH 7.5) containing 1 mM CaCl2 or RPMI 1640 medium containing 10% fetal calf serum and 5 µg/ml gentamicin
(complete medium). For the generation of macrophages, the mononuclear
cell preparation in complete medium was incubated for 2 h in
plastic flasks and then gently washed to remove non-adherent cells. The
plastic-adherent monocytes were differentiated into macrophages by
culturing for 7 days in complete medium. Macrophages were activated by
adding 100 units/ml interferon- 86Rb+ Efflux
Measurements--
ATP-induced 86Rb+ efflux
from chronic lymphocytic leukemia (CLL) lymphocytes was performed as
described (25).
Ba2+ Influx Measurements--
Mononuclear cells
(4 × 106) were incubated with Fura Red (1 µg/ml)
for 30 min at 37 °C in HEPES-buffered NaCl medium. Cells then were
washed once and labeled with appropriate FITC-conjugated anti-CD mAb
for 15 min. Cells were washed once and resuspended in 1.0 ml of
HEPES-buffered KCl medium (150 mM KCl, 10 mM
HEPES, 5 mM D-glucose, 0.1% bovine serum
albumin, pH 7.5) at 37 °C. All samples were stirred and maintained
at 37 °C using a Time Zero module (Cytek, Fremont, CA).
BaCl2 (1.0 mM) was added and was followed
40 s later by the addition of 1.0 mM ATP. Cells were analyzed at 2000 events/s on a FACSCalibur flow cytometer (BD Biosciences) and were gated by forward and side scatter and by cell
type-specific antibodies. The linear mean channel of fluorescence intensity (0-1023 channel) for each gated subpopulation over
successive 2-s intervals was analyzed by WinMDI software (Joseph
Trotter, version 2.7) and plotted against time.
Ethidium+ Influx Measurements--
ATP-induced
ethidium+ influx into mononuclear cells from normal and CLL
subjects and into HEK-293 cells was performed using time-resolved flow
cytometry as described (22). Because of the increased P2X7
function on macrophages, data for comparison of ethidium+
uptake between monocytes and macrophages (Fig. 6) were acquired at a
reduced voltage setting for FL-2 (ethidium+
fluorescence) as described (7).
DNA Extraction, Polymerase Chain Reaction, and DNA
Sequencing--
Genomic DNA was extracted from peripheral blood using
the Wizard genomic DNA purification kit (Promega) according to the
manufacturer's instructions. A primer pair was designed within exon 13 of the P2X7 gene to amplify a 579-bp product from genomic
DNA. P2X7 oligonucleotides were synthesized by Invitrogen.
The forward primer was 5'-GAACCTAGAACCTGAGGGCT-3', and the reverse
primer was 5'-CAGACGTGAGCCACGGTGC-3'. PCR amplification (32 cycles of denaturation at 95 °C for 45 s, annealing
at 56 °C for 45 s, and extension at 72 °C for 1 min)
produced a fragment of the expected 579-bp size. Amplified PCR products
were purified (GFXTM PCR DNA and gel band purification kit,
Amersham Biosciences) and sequenced as described (20).
Site-directed Mutagenesis--
Mutated 1729T Transfection of HEK-293 Cells--
HEK-293 cells were
transfected with P2X7 cDNA using
LipofectAMINETM 2000 reagent diluted in Opti-MEM I medium
as described (20). Cells were collected after 40-44 h by mechanical scraping.
Immunofluorescent Staining and Flow Cytometry--
Surface and
intracellular P2X7 receptor expression on mononuclear cells
and on HEK-293 cells was measured by multicolor flow cytometry as
described previously (22).
Immunofluorescent Staining and Confocal Microscopy--
HEK-293
cells incubated on collagen-coated coverslips were fixed (2%
paraformaldehyde), and P2X7 receptor expression was
determined using immunocytochemistry and confocal microscopy as
described (26). For intracellular staining, cells were permeabilized
with 0.1% Triton X-100, 0.1% dimethyl sulfoxide, and 2% normal horse serum in phosphate-buffered saline for 10 min and washed three times
with phosphate-buffered saline before immunolabeling.
A Single Nucleotide Polymorphism at Position 1729 of the P2X7
Gene--
A PCR product was amplified directly from
genomic DNA to include the whole of exon 13 of the
P2X7 gene, and the product was sequenced. In three of
85 normal subjects, a heterozygous nucleotide substitution (thymine to
adenine) was found at position 1729, but no homozygous substitutions
were observed. The overall allele frequency of this single nucleotide
polymorphism (1729T ATP-induced 86Rb Efflux from Lymphocytes--
The
function of the P2X7 channel was measured by the
ATP-induced efflux of isotopic Rb+ from lymphocytes (>98%
purity) from the peripheral blood of subjects with CLL (25). The
lymphocytes were loaded with 86Rb+ in a 2-h
preincubation, washed, and incubated at 37 °C with or without the
addition of 1 mM ATP. In lymphocytes that were wild type
for both 1729T and 1513A alleles, the loss of
86Rb+ from the cells over 4 min followed
first-order kinetics with a rate constant of 0.03 ± 0.01 min P2X7 Expression and Function in Lymphocytes and
Monocytes--
The permeability of the P2X7 channel was
studied by two-color flow cytometry in which the influx of
Ba2+ was measured into lymphocytes (either T-lymphocyte or
NK cell subsets) identified by appropriate FITC-conjugated mAb.
Fig. 2 shows the ATP-induced uptake of
Ba2+ into lymphocytes from a normal subject loaded with
Fura Red for which fluorescence emission measured by flow cytometry
decreases upon the entry of Ba2+ into the cell. The
rate of Ba2+ uptake into lymphocytes from this normal
subject heterozygous for the 1729A allele was markedly decreased
compared with a normal subject who was wild type at this position (Fig.
2). Similar results were observed for the two CLL subjects heterozygous
for the 1729A allele (data not shown). A more quantitative estimate of
P2X7 receptor channel function was obtained by ATP-induced
uptake of a larger permeant, the fluorescent cation
ethidium+, for which uptake into lymphocyte and monocyte
subsets was measured by time-resolved two-color flow cytometry (22). In
three normal subjects who were heterozygous for 1729A, the ATP-induced
ethidium+ uptakes were extremely low (only 11-26% of
normal mean values (Table I)). The
magnitude of the reduction was similar and significant for the three
normal cell types studied (T-lymphocytes, p < 0.05; B-lymphocytes, p < 0.01; monocytes, p < 0.02; Mann-Whitney U test). Surface expression of the
P2X7 receptor was measured by the binding of a
FITC-conjugated mAb against the extracellular domain of
P2X7 (21). P2X7 expression in the three normal
subjects heterozygous for the 1729A allele averaged 35 and 50% of the
normal mean expression on T- and B-lymphocytes, respectively. Similar
results were obtained for lymphocytes and monocytes from two of the
subjects with CLL who were heterozygous for the 1729A allele. Both had
very low or absent P2X7 function in T-lymphocyte,
B-lymphocyte, and monocyte subsets, whereas the surface expression of
P2X7 in one heterozygous CLL subject was less than half of
the mean value for wild type cells from normal subjects (Table I).
Expression and Function of Ile-568 to Asn-mutated
P2X7--
cDNA for wild type P2X7 or
P2X7 carrying the 1729T Macrophage P2X7 Function--
Differentiation of
monocytes into macrophages increases the expression and function of
P2X7 by manyfold (27). Peripheral blood monocytes were
cultured for 7 days (with interferon- The present study has identified a single nucleotide
polymorphism that leads to a loss of function of P2X7
because of a trafficking defect in this receptor. This polymorphism was
at position 1729T Wide variations in the function of the P2X7 receptor have
been observed in lymphocytes both from normal subjects (20) and patients with CLL (30). Some but not all of this variability is a
result of a single nucleotide polymorphism (1513A An increasing number of channelopathies have been associated with human
disease. Thus hypokalemic periodic paralysis and familial hemiplegic
migraine result from missense or point mutations in a voltage-sensitive
calcium channel (33, 34). Mutations in sodium and potassium channels
also can cause neurological disease with ataxic or epileptic phenotypes
(34-36). There is increasing interest in channelopathies that affect
cells of the immune system and the impact of reduced or absent
P2X7 channel function on human susceptibility to infectious
diseases. P2X7 receptor expression is up-regulated on
differentiation of monocytes to macrophages (27), where its activation
is necessary both for the ATP-induced killing of ingested mycobacterial
species and for the subsequent apoptotic death of this phagocytic cell
(37-39). Whether Asn-568 (1729A allele) as well as Ala-496 (1513C
allele) contribute to the genetic susceptibility to tuberculous
infection is uncertain, although macrophages from subjects heterozygous
for Asn-568 failed to up-regulate P2X7 function to the same
extent as wild type subjects (Fig. 6). Although a mouse strain of
P2X7-null genotype has been developed, there is no
distinctive adverse phenotype of this animal (40, 41). However, the
severity of inflammatory arthritis induced by anti-collagen antibody
was markedly attenuated in these animals (41). It seems likely that the
P2X7 receptor and its polymorphic variants will be central
in our understanding of certain inflammatory and infectious diseases.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C), present in around 20% of the population, which changes Glu to Ala at amino acid 496 and which leads to loss of function of the
receptor (20). Surface expression of P2X7 on lymphocytes was not affected by the Glu-496 to Ala polymorphism, suggesting that
the loss of function resulted from impaired protein-protein interactions in the P2X7 complex at the cell membrane
rather than from trafficking to the surface (20). In this study, we
report the functional effects of a second polymorphism of the human
P2X7 gene (thymine to adenine at position 1729 of cDNA)
that is associated with loss of function of the P2X7
receptor because of failure of its trafficking to the cell surface.
This polymorphism changes Ile to Asn at amino acid 568, which localizes
this residue as being within a trafficking motif in the carboxyl tail
of the receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was obtained from Roche
Diagnostics. HEPES, fetal calf serum, normal horse serum,
LipofectAMINETM 2000 reagent, Opti-MEM I medium, and
Taq DNA polymerase were purchased from Invitrogen.
Ficoll-PaqueTM and 86RbCl (1.5 mCi/ml; specific
radioactivity, 3 Ci/mmol) were purchased from Amersham
Biosciences. Di-n-butyl phthalate and di-isooctyl phthalate (Merck Chemicals Ltd., Poole, England) were blended 80:20
(v/v) to give a mixture of density 1.030 g/ml. Fura RedTM,
AM was obtained from Molecular Probes. Fluorescein
isothiocyanate (FITC)- and phycoerythrin-conjugated anti-CD monoclonal
antibodies (mAb) were acquired from Dako. Cy3-conjugated donkey
anti-sheep IgG antibody was purchased from Jackson ImmunoResearch.
Murine anti-human P2X7 receptor mAb (kindly provided by
Drs. Gary Buell and Ian Chessell) (21) was purified from clone L4
hybridoma supernatant by chromatography on protein A-Sepharose fast
flow and conjugated to FITC as described (22). Sheep polyclonal
anti-P2X7 antibody against a non-homologous extracellular
epitope of the human P2X7 receptor (23) was produced as
described for a sheep polyclonal anti-P2Y1 antibody (24).
Specificity of the polyclonal anti-P2X7 antibody was
confirmed by binding to human embryonic kidney (HEK)-293 cells
transfected with human P2X7 cDNA but not to
mock-transfected or non-transfected HEK-293 cells (Fig. 5 and results
not shown). Preimmune serum failed to bind transfected, mock-transfected, or non-transfected HEK-293 cells (results not shown).
in the final 24 h of culture
before harvesting by mechanical scraping for flow cytometric analysis.
A was introduced
using overlap PCR (QuikChangeTM site-directed mutagenesis
kit, Stratagene) and the expression vector pCI-hP2X7 as
described (20). The primer sequences were as follows (base changes are
in bold and underlined): T1729A forward, C ATG GCT GAC TTT GCC AAC CTG CCC AGC TGC TGC C;
T1729A reverse,
G GCA GCA GCT GGG CAG GTT GGC AAA GTC AGC CAT G.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A) was 0.04 in the Caucasian population. A
comparable allele frequency of 0.06 was found in 45 patients with CLL
of whom three were heterozygous for the 1729A allele. The deduced amino
acid change for this polymorphism is isoleucine to asparagine at amino
acid 568 (I568N) of the P2X7 protein. All six subjects who
were heterozygous for 1729T
A were wild type for the polymorphism
that we have described previously at nucleotide 1513 of the
P2X7 gene (20). Subjects identified previously as
homozygous for the loss-of-function 1513A
C polymorphism (20) were
wild type at position 1729.
1 (range of 0.01-0.05, n = 7) in the
absence of ATP and 0.34 ± 0.04 min
1 (range of
0.24-0.50, n = 7) in the presence of ATP (Fig.
1). In two subjects heterozygous for the
1729A allele, the function of the lymphocyte P2X7 channel
was either absent or reduced (Fig. 1). In one heterozygote the rate
constant for 86Rb+ efflux from lymphocytes was
the same in the absence and presence of ATP (0.02 and 0.03 min
1, respectively), whereas in the other heterozygote
the addition of ATP increased the rate constant from 0.02 to 0.23 min
1. Data from duplicate experiments for each
heterozygote provided in the presence of ATP a mean rate constant of
0.12 ± 0.07 min
1 (n = 4), which was
significantly lower than the rate constant of 0.34 ± 0.04 min
1 (n = 7) for wild type lymphocytes
(p < 0.01, Mann-Whitney U test). Measurement of isotopic Rb+ efflux from lymphocytes of
normal subjects was complicated by the variable admixture of monocytes
with lymphocytes, and we turned to flow cytometric methods to study
P2X7 channel fluxes in defined leukocyte subsets.
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Fig. 1.
ATP-induced Rb+ efflux from
lymphocytes from CLL subjects either wild type or heterozygous for the
1729A allele. Lymphocytes from CLL subjects either wild type at
1729 ( ) or heterozygous for the 1729A allele (
,
) were loaded
for 2 h with 86Rb+. Cells were washed,
resuspended in KCl medium, and incubated for 5 min at 37 °C before
incubation with 0.5 mM ATP for 4 min. Samples (1 ml) were
collected at 1-min intervals. Basal 86Rb+
efflux measured in the absence of ATP is shown (
).
86Rb+ efflux is expressed as (1
Nt/No), where
Nt is the level of cell-associated radioactivity
at time t (determined by Cerenkov counting) and
No the amount of cell-associated radioactivity
at time 0. The data have been analyzed after log transformation
to permit calculation of efflux rate constants. Results from seven
different subjects wild type at 1729 are shown as the mean ± S.E.
Results from two different subjects heterozygous for the 1729A
allele are shown as separate lines (one line
being representative of two experiments for each heterozygote
subject).
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Fig. 2.
ATP-induced Ba2+ influx into
normal human T-lymphocytes and NK cells from subjects either wild type
or heterozygous for the 1729A allele. Mononuclear cells from
normal subjects either wild type at 1729 ( ) or heterozygous for the
1729A allele (
) were incubated with 1 µg/ml Fura Red for 30 min
and washed once. The cells were labeled with FITC-conjugated
anti-CD3 or -CD16 mAb and resuspended in KCl medium at 37 °C.
Ba2+ (1 mM) was added and was followed 40 s later by the addition of 1 mM ATP (arrow). The
mean channel of cell-associated fluorescence intensity was measured at
2-s intervals for gated CD3+ T-lymphocytes (top
panel) or CD16+ NK cells (bottom panel).
Basal Ba+ influx measured in the absence of ATP is shown
(
). The arbitrary units of the area above the Ba2+
influx curve in the first 20 s after the addition of ATP
are 1519 for wild type T-lymphocytes, 720 for heterozygous
T-lymphocytes, 2265 for wild type NK cells, and 1038 for heterozygous
NK cells. The figure depicts one representative experiment of three
performed.
Reduced P2X7 expression and function in leukocytes with the
1729A allele
A mutation was transfected into
HEK-293 cells, and the expression and function of the receptor were
measured. At 40 h after transfection, strong surface expression
and function of wild type P2X7 were observed, but the
1729T
A-mutated P2X7 was non-functional using the
ATP-induced ethidium+ uptake assay (Fig.
3). The basis for the loss of
P2X7 function was failure of trafficking of the mutant
receptor to the cell surface shown by the absence of surface
P2X7 immunoreactivity in cells transfected with mutated
P2X7 and incubated with a mAb to the extracellular domain
of P2X7 (Fig. 4). Confocal
microscopy confirmed the absence of surface expression of
1729T
A-mutated P2X7, although strong intracellular
staining for mutated P2X7 was seen in permeabilized cells
(Fig. 5).
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Fig. 3.
ATP-induced ethidium+ uptake into
HEK-293 cells transfected with wild type or 1729A-mutated
P2X7 cDNA. HEK-293 cells transiently transfected
either with wild type ( ) or 1729A-mutated P2X7 cDNA
(
) or with non-transfected HEK-293 cells (
) were washed and
resuspended in 1 ml of KCl medium. Ethidium+ (25 µM) was added and was followed 40 s later by the
addition of 1 mM ATP (arrow). The mean channel
of cell-associated fluorescence intensity was measured at 5-s intervals
for gated HEK-293 cells. The figure depicts one representative
experiment of three performed.
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Fig. 4.
Flow cytometric histograms of wild type and
1729A-mutated P2X7 expression in HEK-293 cells.
HEK-293 cells were transiently transfected with either wild type
(left panels) or 1729A-mutated (right panels)
P2X7 cDNA. Non-permeabilized cells (top
panels, surface P2X7 expression) and fixed and
permeabilized cells (bottom panels, intracellular
P2X7 expression) were labeled with FITC-conjugated
anti-P2X7 mAb (black line) or isotype control
mAb (shaded line), and the level of P2X7
expression was determined by flow cytometry. The figure depicts one
representative experiment of three performed.
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Fig. 5.
Confocal microscope images of wild type and
1729A-mutated P2X7 expression in HEK-293 cells.
HEK-293 cells were transiently transfected with either wild type
(left panels) or 1729A-mutated (right panels)
P2X7 cDNA. Non-permeabilized (top
panels, surface P2X7 expression) or permeabilized
(bottom panels, intracellular P2X7 expression)
cells were incubated with anti-P2X7 antibody and
subsequently with Cy3-conjugated anti-sheep IgG antibody before
analysis by confocal microscopy. The calibration bar is 2 µm.
present for the final 24 h), and the P2X7 function was measured by ATP-induced ethidium+ uptake into the CD14+ macrophage
population. Macrophages from a normal subject with wild type
P2X7 had an ATP-induced ethidium+ uptake
10-fold greater than their precursor monocytes (Fig.
6). Thus the area under the ATP-induced
ethidium+ uptake curve increased from 5193 units on day 0 monocytes to 49,314 units on day 7 macrophages. Macrophages from a
normal subject heterozygous for the 1729A allele also increased their
P2X7 function from 845 units on day 0 monocytes to 26,606 on day 7 macrophages, but this function was only 54% that of
macrophages that were wild type with both 1729T and 1513A alleles (Fig.
6).
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Fig. 6.
ATP-induced ethidium+ uptake in
monocytes and monocyte-derived macrophages from normal subjects either
wild type or heterozygous at position 1729 of the P2X7
gene. Fresh monocytes ( ,
) or 7-day monocyte-derived
macrophages (activated with interferon-
) (
,
) from subjects
either wild type at 1729 (
,
) or heterozygous for the 1729A
allele (
,
) were labeled with FITC-conjugated anti-CD14 mAb and
suspended in KCl medium at 37 °C. Ethidium+ (25 µM) was added and was followed 40 s later by the
addition of 1 mM ATP (arrow). The mean channel
of cell-associated fluorescence was measured by time-resolved flow
cytometry. Basal ethidium+ uptake measured in the absence
of ATP is shown (
). The figure depicts one representative experiment
of two performed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A of the cDNA that changes isoleucine to
asparagine at amino acid position 568. Three of 85 normal subjects were
found to be heterozygous for this polymorphic variation, and all three
had half-normal expression of the P2X7 on the cell surface
and reduction of P2X7 function to ~25% of normal.
Transfection experiments provided confirmation that the Ile-568 to
Asn-mutated P2X7 fails to traffic to the cell surface.
HEK-293 cells transfected with this mutated P2X7 showed
neither surface expression nor function despite plentiful intracellular
synthesis of this receptor (Figs. 4 and 5). In other experiments, we
measured the physiological properties of the Ile-568 to Asn-mutated
P2X7 in Xenopus oocytes and found channel
currents in oocytes that were identical to those with the wild type
P2X7 construct.2
This result shows that the Ile-568 to Asn mutation does not alter the
function of the P2X7 channel, although this result shows
differences in trafficking between the mammalian and amphibian
expression systems. A number of functional domains have been proposed
in the long carboxyl terminus of P2X7 based on protein
sequence homology. The most distal of these, the "LPS-binding
domain" from amino acids 573 to 590, has homology with the
LPS-binding protein of plasma and has been suggested to interact
directly with internalized LPS (16). Using a series of truncated
mutants of rat P2X7, we have identified previously a region
between residues 551 and 581 that is both highly conserved and
necessary for the surface expression of this receptor (19). Mutation of
the conserved residues at Cys-572, Arg-574, and Phe-581 abolished both
cell surface expression and receptor function (19), suggesting that the
trafficking domain of P2X7 overlaps the putative
LPS-binding domain. The present data add the conserved Ile-568 to the
other residues within this trafficking domain required for cell surface
expression of P2X7. Whether this domain binds directly to
phospholipid or to one of the many protein partners in the
P2X7 membrane complex (15) is uncertain. However, this
domain includes a conserved cysteine, palmitoylation of which is
required for cell surface expression in a number of other receptors
such as CCR5 (28). Recently, P2X4 but not P2X2
receptors have been shown to undergo constitutive endocytosis in
neurons (29). Whether P2X7 also undergoes constitutive endocytosis is unknown, but the lack of 1729T
A-mutated
P2X7 expressed on the surface of HEK-293 cells may be
because of the rapid endocytosis of the receptor after initial
trafficking to the cell surface. This, however, seems unlikely, as we
failed to detect mutant P2X7 on the surface of HEK-293
cells using either flow cytometry or confocal microscopy.
C) that changes
Glu-496 to Ala and leads to loss of function without affecting surface
expression of the receptor in lymphocytes (20). The trafficking-defective polymorphism identified in the present study, Ile-568 to Asn, contributes to this variability in P2X7
function but with a lower heterozygote prevalence of ~4% compared
with ~20% for the more common Glu-496 to Ala variation.
Collectively, however, these two genotypes still do not account for all
individuals with low or absent P2X7 function (20, 30). The
coding region within exon 13 of the P2X7 gene is highly
polymorphic with five single nucleotide polymorphisms identified within
a 0.5-kb stretch of
cDNA,3 and it is likely
that other loss-of-function polymorphisms exist in the P2X7
gene, both within the coding region and in the upstream promoter of
this gene (31). Recently a polymorphism affecting the function of the
mouse P2X7 receptor has been recognized in inbred strains
of mice. Although most strains of mice carried Pro-451 with good
P2X7 function, C57BL and DBA strains possessed a Leu-451
allele with lower P2X7 function (32). Whether the recognition or docking sequence around residue 451 lies within a domain
interacting with a Src homology 3-binding protein is not known. We have
surveyed 99 human base sequences coding for residue 451 but have found
no deviations from the wild type
sequence.4
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ACKNOWLEDGEMENTS |
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We thank Dr. Diane Williams for blood collection; Prof. Graeme Stewart, Dr. David Booth, and Maria Ban for helpful discussions; Shelley Spicer for typing this manuscript; and Kristen Skarratt for critically reviewing the manuscript.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council, the Cure Cancer Australia Foundation, and the Leukaemia Foundation of Australia.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: Level 5, South Block, Nepean Hospital, Penrith, NSW 2750, Australia. Tel.: 61-2-4734-3277; Fax: 61-2-4734-3432; E-mail: wileyj@medicine.usyd.edu.au.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M212759200
2 M. L. Smart, unpublished observations.
3 C. Li, R. Sluyter, B. J. Gu, and J. S. Wiley, unpublished observations.
4 C. Li and J. S. Wiley, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: LPS, lipopolysaccharide; CLL, chronic lymphocytic leukemia; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody(ies); HEK, human embryonic kidney; NK, natural killer.
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