From the Departments of 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
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ABSTRACT |
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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 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.
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- 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 1513A 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).
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).
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
1513A The 1513A 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 1513A 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 1513A Function of 1513A 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- 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.
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 1513A The polymorphic 1513A Transfection of 1513A 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
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.
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-
, 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
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Macrophages were then
detached by mechanical scraping for flow cytometric analysis.
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 DH5
cells. All mutations were confirmed by sequencing. The
primer sequences were as follows: 1513A
C (E496A) forward, GG.TGC.CTG.GAG.GCG.CTG.TGC.TGC.CGG; 1513A
C
(E496A) reverse, CCG.GCA.GCA.CAG.CGC.CTC.CAG.GCA.CC. Base changes
introducing the mutations are in bold type and underlined.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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.
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Fig. 2.
Sequence of genomic DNA at the
C-terminal tail of the P2X7 gene showing
(a) germline, (b) 1513A C
homozygous mutant, and (c) 1513A
C
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.
C 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).
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 (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) 1513A C
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.
Expression and function of P2X7 receptor in mononuclear cells
from 20 normal subjects and 1 B-CLL subject with germline and 1513AC
mutant P2X7
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 (12).
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Fig. 4.
Typical ATP-induced ethidium uptake
curve in mononuclear cell subsets from normal subjects with germline,
1513A C heterozygous, and 1513A
C homozygous P2X7 as
indicated. A single patient with B-chronic lymphocytic leukemia
who was 1513A
C 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 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.
C Mutated P2X7 Transfected into
HEK293--
cDNA for germline P2X7 or P2X7
carrying 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.
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 1513A C 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.
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 1513A
C 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 1513A C 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-
. 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.
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Fig. 8.
BzATP-induced cytotoxicity of T-lymphocytes
from subjects with germline and 1513A C 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
C 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 1513A
C 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 1513A
C 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 1513A
C 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 1513A
C mutant P2X7 allele in
homozygous dosage, whereas the other two patients were germline at this
position.3 Clearly 1513A
C
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
-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.
C 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.
C 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-
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.
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
-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.
![]() |
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.
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REFERENCES |
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