From the Department of Physiology, the University of
Melbourne, Victoria 3010, the ¶ Department of Medicine, the
University of Sydney, NSW 2006, and the
St. Vincent's Medical
Institute, Victoria 3065, Australia
Received for publication, October 30, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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The importance of the cytosolic C-terminal region
of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to
the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was
performed in HEK-293 cells and Xenopus oocytes. Cells
expressing receptors truncated at residue 581 (of 595) have negligible
ethidium ion uptake, whereas those expressing the P2X7R truncated at
position 582 give wild type ethidium ion uptake suggesting that pore
formation requires over 95% of the C-terminal tail. Channel function
was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region
between residues 551 and 581 resulted in non-functional receptors with
no detectable cell surface expression in HEK-293 cells. A more detailed
analysis revealed that mutations of single residues within this region
could also abolish receptor function and cell surface expression,
suggesting that this region may participate in regulating the surface
expression of the pore-forming P2X7R.
The P2X7R1 is a
ligand-gated ion channel and the seventh member of the P2X family (1).
Exposure to ATP or the more potent agonist BzATP to the P2X7R renders
the P2X7R permeant to Na+, K+, and
Ca2+ (2). Repeated or prolonged application of either
agonist induces the formation of a cytolytic pore that is permeable to
larger cations such as positively charged ethidium or
N-methyl-D-glucamine (3, 4). The ability
of the P2X7R to form pores is dependent on experimental conditions.
Factors such as the duration of the agonist application, the presence
of divalent cations, extracellular pH, receptor density, and receptor
species influence pore formation (1, 4-6).
The conversion from channel to pore state may reflect either a
conformational change within the receptor's selectivity filter (7) or
the successive oligomerization of additional P2X7R subunits (8). We
recently examined whether the P2X7R formed clusters (9) upon agonist
activation, as a prelude to density-dependent pore
formation. We found that, unlike the P2X2R, which clusters upon agonist
activation (1-2 µm in size) (10), the P2X7R does not exist in
clusters in the basal state or upon receptor activation (9). Although
receptor density regulates pore formation in cells that endogenously
express the P2X7R (6), a localized increase in receptor density or
clusters is not associated with P2X7 pore formation.
The P2X7R shows structural homology with other P2X family members,
consisting of two transmembrane domains connected by a large
extracellular loop. The extracellular loop contains conserved cysteine,
lysine, and glycine residues along with a number of potential
N-linked glycosylation sites, all of which contribute to the
structural constraints required for ATP binding (11-13). The C
terminus of the P2X7R is 120 amino acids longer than any of the other
P2X members. This long C-terminal domain (~240 amino acids) appears
to modulate the function of the P2X7R, because the removal of this
region tempers the receptor's response to ATP. In particular, the
removal of the last 177 amino acids (to yield P2X7-(1-418))
abolishes pore formation without affecting channel properties (1, 14).
Investigation of a 1-439 truncated human P2X7 receptor in
Xenopus oocytes revealed an altered kinetic response to ATP
applications, thought to be related to a difference in the agonist
binding site of the truncated receptor compared with the full-length
P2X7R (14). Furthermore, when the human P2X7R is truncated at residue
415, it does not exhibit cell surface expression (15).
Sequence analysis of the cytosolic C-terminal of the P2X7R has
supported the idea that the C-terminal region is important for receptor
function. Denlinger and colleagues (16) identified a putative
lipopolysaccharide binding domain and went on to corroborate this
sequence analysis with biochemical evidence that this putative domain
actually binds lipopolysaccharide. In a large scale proteomic analysis,
Kim and co-workers (17) identified numerous putative protein partners,
many of which are thought to interact with the C-terminal region. In
particular, epithelial membrane proteins were found to interact with
the C-terminal tail of the P2X7R and are involved in cell blebbing
(18). Clinical investigations have shed further light on the
role of the C-terminal region and have demonstrated that an E496A
polymorphism seen in the human P2X7R is associated with a loss of
function and the appearance of CLL in patients harboring this amino
acid change (19).
Further evidence for a role of the C-terminal in regulating pore
formation comes from studies of rat, human, and mouse P2X7 receptors.
There are differences in the ability of these receptors to form pores,
with the rat P2X7R considered as the "best" (4). Augmented
pore-forming ability can be conferred to the human receptor by
substitution of the human intracellular C-terminal region with the
corresponding region of the rat P2X7R (20), suggesting that residues
within this domain participate in pore formation.
Thus, our accumulated knowledge is consistent with the idea that the C
terminus is an important modulator of receptor function and dysfunction
with significant clinical manifestations. Yet, little is known about
the identity of functional domains of the C terminus that presumably
underlie these important functions. In the present study we sought to
define such functional domains of the C-terminal region by a systematic
study of a series of P2X7 truncations and point mutants. Pore formation
was determined by ethidium ion uptake in HEK-293 cells, and channel
conductance was measured in Xenopus oocytes with the
two-electrode voltage clamp method. We present experimental evidence
that P2X7R pore formation is dependent upon a distal region of the C
terminus, and this region modulates the surface expression of the pore- forming P2X7R.
Molecular Biology
The wild type clone of rat P2X7R (accession number X95882, a
kind gift from Dr. Gary Buell) was used in these studies. Truncated
P2X7 receptors were created using standard PCR protocols. A
HindIII and EcoRI restriction site was
incorporated at the 5' and 3' ends of each PCR product. All PCR
products were subcloned into the pcDNA3.1(+) vector (Invitrogen,
Mount Waverley, Victoria, Australia) and amplified using competent
DH5 A total of seven separate sites were targeted for glycine substitution.
The following residues were chosen for our studies: Cys548, Cys572, Arg574,
Lys576, Glu580, Phe581, and
Pro582 with each residue (in the full-length P2X7R) mutated
to glycine. Site-directed mutagenesis was performed using Stratagene's
QuikChange site-directed mutagenesis kit (Integrated Sciences,
Willoughby, New South Wales, Australia) as per the manufacturer's
instructions, and the forward and reverse primers for each mutant were
designed as recommended. The forward and reverse primers for each
mutant are listed as follows: C548G forward: 5'-C AGC AAG CTG CGA CAC GGT GCG TAC AGG AGC TAT GCC; C548G reverse:
5'-GGC ATA GCT CCT GTA CGC ACC GTG TCG CAG CTT
GCT G; C572G forward: 5'-GCC ATT CTG CCC AGC GGC
TGC CGC TGG AAG ATC CGG; C572G reverse: 5'-CCG GAT CTT CCA GCG GCA
GCC GCT GGG CAG AAT GGC; R574G forward: 5'-CTG
CCC AGC TGC TGC GGC TGG AAG ATC CGG AAG; R574G
reverse: 5'-CTT CCG GAT CTT CCA GCC GCA GCA GCT
GGG CAG; K576G forward: 5'-CC AGC TGC TGC CGC TGG GGG ATC CGG AAG GAG TTC CC; K576G reverse: 5'-GG
GAA CTC CTT CCG GAT CCC CCA GCG GCA GCA GCT GG;
E580G forward: 5'-GG AAG ATC CGG AAG GGG TTC CCC
AAG ACC CAG; E580G reverse: 5'-CTG GGT CTT GGG GAA
CCC CTT CCG GAT CTT CC; F581G forward: 5'-GG AAG
ATC CGG AAG GAG GGC GGG AAG ACC CAG GGG; F581G reverse: 5'-CCC CTG GGT CTT GGG GCC CTC CTT CCG
GAT CTT CC; P582G forward: 5'- G ATC CGG AAG GAG TTC
GGC AAG ACC CAG GGG CAG; P582G reverse: 5'-CTG
CCC CTG GGT CTT GCC GAA CTC CTT CCG GAT C. Base
changes introducing the mutations are in boldface type and underlined.
Three mutants, Cys572 For electrophysiological studies, the truncated receptor constructs
were linearized with XbaI in preparation for transcription run-off. In vitro transcription was performed using
Ambion's T7 mMessage mMachine mRNA kit (GeneWorks, Thebarton,
South Australia, Australia) as per the manufacturer's
instructions. The full-length, capped cRNAs were purified using
diethyl-pyrocarbonate H2O-equilibrated Clontech Chroma Spin-100 columns (Progen
Industries, Brisbane, Australia). cRNA samples were electrophoresed on
a denaturing agarose gel to determine the quantity, quality, and size
of the transcript.
Cell Culture
HEK-293 and COS-7 cells were grown at 37 °C, 5%
CO2 in Dulbecco's modified Eagle's medium, supplemented
with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 units/ml). For ethidium ion uptake experiments HEK-293 cells were
dislodged using Trypsin-EDTA and seeded into 96-well plates, then
transfected 24 h later (or when confluency had reached ~75%).
30 µl of Effectene buffer, 0.8 µl of enhancer, 2.5 µl of
Effectene reagent (Qiagen, Clifton Hill, Victoria, Australia) were
assembled with 0.8 µg of sample DNA. This mixture was used to
transfect eight wells. Following a 12-h period the transfection reagent
was replaced with fresh medium. For imaging studies, the HEK-293 and
COS-7 cells were seeded onto poly-L-lysine-treated
coverslips. Cells were transfected with the wild type rat P2X7-EGFP,
Cys572 Ethidium Ion Uptake
Pore formation was examined in HEK-293 cells transiently
transfected with the wild type rat P2X7R, truncated P2X7 receptors, and
C-terminal mutants. A plate reader (Wallac 1420, PerkinElmer Life
Sciences) was used to determine ethidium ion uptake in these cells.
Three days post-transfection the cells were washed once with low
divalent HEPES buffer (in millimolar; NaCl 147, KCl 2, HEPES 10, glucose 10, CaCl2 0.1, pH 7.4). Basal ethidium ion
fluorescence was assessed once the wash buffer was replaced with low
divalent HEPES supplemented with ethidium bromide (10 µg/ml).
Fluorescence signals from ethidium ion were excited with the 485 ± 15-nm band of a 75-watt tungsten-halogen lamp, and emission greater
then 615 ± 8.5 nm was collected. Once background ethidium ion
fluorescence was measured, this "ethidium only" solution was
replaced with low divalent HEPES supplemented with BzATP (100 µM) and ethidium bromide (10 µg/ml). Ethidium ion
fluorescence was measured at four separate locations within each well,
and an entire plate was read ten times within a 30-min period. All
ethidium ion uptake experiments were carried out at 37 °C.
Confocal Microscopy
Calcium Uptake--
HEK-293 cells were loaded with fluo-4 AM for
20 min at 37 °C then washed twice before experiments. Cells were
illuminated with the 488-nm line of a 100-milliwatt argon ion laser and
viewed on an MRC-1024 (Bio-Rad, Regents Park, New South Wales,
Australia) confocal microscope. Data was collected using the
single-green fluorophore emission filter set. Fluorescence signals from
fluo-4 were sampled every 3 s and collected using a software
module (Laser Sharp Timecourse, version 3.1) for a period of 5 min.
Recordings were made in high divalent HEPES buffer (in millimolar; NaCl
147, KCl 2, HEPES 10, glucose 10, CaCl2 1, pH 7.4),
supplemented with BzATP (50 µM). The fluo-4 fluorescence
intensity recorded over a 5-min period was integrated to give an index
of channel activity. This provided an estimation of the amount of
calcium entry through the P2X channel versus the calcium
release from internal stores by P2Y activation.
Receptor Localization--
Transfected cells were illuminated
with the 488-nm line of a 100-milliwatt argon ion laser and viewed on a
Bio-Rad MRC-1024 confocal microscope. Fluorescence signals from EGFP
were viewed while cells were perfused with a low divalent HEPES buffer.
Electrophysiology
Oocytes from adult female Xenopus laevis
were surgically removed and prepared as outlined previously (21). Stage
5 or 6 oocytes were injected with cRNA-encoding wild type rat P2X7R, truncated P2X7 receptors, or the C-terminal mutants and were stored at
18 °C for 3 days prior to experimentation. For two electrode voltage
clamp recordings, oocytes were impaled with two glass electrodes
containing 3 M KCl and held at a membrane potential of
Flow Cytometry
HEK-293 cells were cultured in RPMI 1640 media supplemented with
10% FBS, 2 mM glutamine, and 0.02 mg/ml gentamicin.
75-cm2 flasks of HEK-293 cells (~90% confluency) were
transfected with 15 µg of the wild type rat P2X7R, truncated P2X7
receptors, or the C-terminal mutants, using the LipofectAMINE 2000 reagent protocol (Invitrogen). After 40-44 h, cells were collected by
mechanical scraping. Cell suspensions (90 µl of 5 × 106 cells/ml in RPMI 1640, 10% FBS) were then incubated
with human group AB serum (10 µl), 7-aminoactinomycin D (20 µg/ml),
and FITC-conjugated mouse anti-human P2X7 monoclonal antibody (clone
L4, 60 µg/ml) (22) at room temperature (~22 °C) for 20 min,
before washing with PBS (pH 7.2). Labeled cells were resuspended in 1 ml of PBS and analyzed on a BD Biosciences FACSCalibur flow cytometer
(San Jose, CA), as previously described (19). The isotype control was a
murine FITC-conjugated IgG2b (DAKO-Cytomation, Glostrup, Denmark).
Values were normalized to the absolute value of expression of the wild
type P2X7R surface expression and corrected for the vector alone control.
Data Analysis
Electrophysiological analysis of two electrode voltage
clamp results was performed using CLAMPFIT module of P-Clamp 8.1 (Axon Instruments, Forster City, CA) and ORIGIN 6.0 (Microcal Software, Northampton, MA). Data in the text and graphs are shown as means ± S.E., and statistical analysis was performed using a one-way ANOVA
(Tukey, family error rate of 0.05). The number of wells, cells, or
oocytes examined for each experimental protocol is presented in parentheses.
Functional Analysis of Pore Formation and Channel Activity in
Truncated Receptors--
A series of truncated P2X7 receptors was
constructed and subjected to functional analysis so that the functional
domains of the intracellular C-terminal domain could be identified
(Fig. 1). Truncated P2X7 receptors were
tested for their ability to form ethidium ion permeant pores using a
plate reader assay designed for rapid screening of transfected HEK-293
cells. Cells transfected with the wild type P2X7R or vector alone were
also assayed for ethidium ion uptake following addition of 100 µM BzATP (Fig.
2A). The wild type receptor
gave a robust and highly reproducible increase in ethidium ion
fluorescence that developed over a period of 30 min. The relative
fluorescence intensity at this time point was chosen for all subsequent
analyses of ethidium ion uptake. Plate reader analysis of ethidium ion
uptake of truncated receptors revealed a strong relationship between
the truncation position and ethidium ion uptake (Fig. 2B).
Under no circumstances could we detect ethidium ion fluorescence for
any receptor that was truncated upstream of residue 582. By contrast,
all constructs that were 582 residues or longer in length displayed
ethidium ion fluorescence levels identical to that of the full-length
receptor. Thus, removal of one residue, proline 582, resulted in a
switch from wild type ethidium ion uptake to complete absence of
uptake. To examine this further we substituted this proline with
glycine, and ethidium ion uptake was re-assessed (Fig. 2B).
Interestingly, this 1-Pro582Gly truncation mutant displayed a level of
ethidium ion uptake that fell almost halfway between the levels
measured with the 1-582 truncation mutant and the full-length
receptor.
The channel properties of truncated receptors were then investigated in
transfected HEK-293 cells loaded with the Ca2+-sensitive
dye, fluo-4 AM, under ionic conditions that would not favor pore
formation (2). Calcium influx was determined by the change in fluo-4
fluorescence upon the addition of 50 µM BzATP (Fig.
2C). The application of BzATP to HEK-293 cells transfected with the pcDNA3.1(+) vector caused an increase in the intracellular calcium concentration, presumably by the release of calcium from intracellular stores via activation of endogenously expressed P2Y
receptors (Fig. 2C). This increase in calcium by P2Y
receptors upon BzATP application has been previously reported for
native HEK-293 cells (23). The addition of 50 µM BzATP to
HEK-293 cells transfected with the full-length rat P2X7R caused a
sustained increase in fluo-4 fluorescence, which was interpreted as an
influx of calcium through the P2X7 channel (Fig. 2C).
Reduction of this type of data by mathematical integration of the
Ca2+ levels over time was a good discriminator of the
difference between a sustained influx and the transient, P2Y-elicited
store release of Ca2+. Furthermore, it correlated well with
some whole cell current recordings made by patch clamp (data not shown)
validating this approach as a rapid method for analysis of P2X7R
channel properties. Analysis of truncated receptors showed that a
sustained increase in Ca2+ was observed in transfected
HEK-293 cells with the constructs, 1-380, 1-400, 1-418, 1-460,
1-500, 1-540, 1-550, 1-582, and 1-Pro582Gly (Fig. 2D),
whereas the following constructs failed to produce a sustained increase
in Ca2+ influx: 1-360, 1-560, 1-570, 1-580, and 1-581
(Fig. 2D). In contrast to pore function, channel function
was clearly evident in many of the shorter truncation constructs.
We next investigated whether the loss of P2X7R channel function seen in
certain truncated receptors may be associated with a decrease in
plasmalemma expression. Cell surface expression was determined using
flow cytometry with the L4 human P2X7 antibody (19). This
FITC-conjugated antibody recognizes an extracellular epitope on the
human P2X7R. HEK-293 cells were transfected with each of the truncated
P2X7 receptors. Two days post transfection, cells were suspended in PBS
and P2X7R surface expression was quantified using a FACSCalibur flow
cytometer. Clear differences in cell surface expression were observed,
with expression levels varying from undetectable to twice that of wild
type receptor. For the most part cell surface expression correlated
well with channel function seen in Fig. 2D with two notable
exceptions, the 1-540 and 1-550 truncations, which displayed
significant channel function but had negligible surface expression.
Clearly, function requires cell surface expression, and this may
represent a limitation of the FACS technique in studying mutant
receptors where differences in epitope presentation and the consequent
reduced antibody binding may be incorrectly interpreted as a lack of
surface expression. The other noteworthy result is that the 1-582
truncation displayed almost twice the level of cell surface expression
compared with that of control. Interestingly, activation of this
truncation gave a robust Ca2+ response (Fig.
2D). In all cases, truncated receptors that displayed a loss
of both channel and pore function could not be detected at the cell
surface (Fig. 2D). As a further check for cell surface expression N- and C-terminal GFP-tagged 1-418, 1-570, and 1-580 truncation constructs were made and examined using confocal microscopy (data not shown). Although neither of the N- and C-terminal-tagged GFP
truncations displayed surface expression, a fluorescence signal was
detected in the cytoplasm, indicating that these receptors were being
translated and expressed.
The channel properties of the truncated receptors were also examined in
Xenopus oocytes. In most cases, the application of 1 mM ATP caused a rapid current (I1) followed by
a delayed current (I2; Fig.
3A) and again correlated quite
well with the Ca2+ influx data shown in Fig. 2D.
As would be expected from the data, in HEK-293 cells negligible current
responses were observed with the 1-360, 1-570, and 1-580 truncations
(Fig. 3B). Two interesting exceptions are truncations
1-560 and 1-581, which showed no significant channel activity in
HEK-293 cells (Fig. 2D) but gave robust current responses in
Xenopus oocytes (Fig. 3B). The
I1 and I2 components of current development
were affected differently by the same truncation, suggesting the
mechanisms responsible for these two distinct functional phases may
also be structurally distinct.
Fig. 3B summarizes the findings above and presents in a
clear fashion several key observations: 1) Truncation between residues 551 and 581 results in a complete abolition of ethidium ion uptake, channel function, and absence of cell surface expression. 2) Truncation upstream results in restoration of cell surface expression and channel
function to varying degrees but significantly above control. 3) The
ability of the receptor to accumulate ethidium ions is not similarly
restored. 4) Truncation at residue 380 results in a receptor that can
still function as an ion channel. Thus, the region between 551 and 581 appears to regulate cell surface expression in conjunction with pore
formation, defined by the ability to rapidly accumulate ethidium ions
upon application of BzATP.
Functional Analysis of Point Mutations of the Distal Region of
C-terminal of the Full-length P2X7R--
To further elucidate the role
of the 551-581 region, single residues within this domain were
targeted for glycine mutagenesis. We also targeted a cysteine residue
at position 548, close to the extremity of the region to examine its
role in receptor function. A phenylalanine at position 581 was
replaced with glycine in an attempt to elucidate why a normal channel
response was observed in oocytes injected with the 1-581 truncation,
yet no functional response was seen with this mutant when expressed in
HEK-293 cells. The proline residue at position 582 was changed to
glycine to investigate whether the reduced ethidium ion uptake observed
with the 1-Pro582Gly truncation indicates that this position plays a
role in the function of the receptor. Thus, two cysteines
(Cys548 and Cys572), two positively charged
residues (Arg574 and Lys576), and three
residues adjacent to the site of truncation where pore formation was
abruptly abolished (Glu580, Phe581, and
Pro582) were chosen for more detailed structure function
analysis (Fig. 1).
The ability of the C-terminal point mutants to accumulate ethidium ions
in response to BzATP was tested in transfected HEK-293 cells using the
plate reader assay described above. Three mutants, Cys572
Channel function was then determined using the fluo-4 assay as
described above. Interestingly, the Cys572
Cell surface expression of the non-functional C-terminal mutants was
examined to determine whether loss of function was related to assembly
or trafficking. Cell surface expression of the C-terminal mutants in
HEK-293 cells was determined using flow cytometry with the L4 human
P2X7 antibody (Fig. 4B). Analysis revealed that mutant receptors that displayed wild type pore formation and channel activity
had similar levels of cell surface expression to the wild type rat
P2X7R (Fig. 4B). Mutant receptors that displayed a loss of
both channel and pore function did not have any cell surface expression
(Fig. 4B).
To further examine the intracellular fate of these non-functional rat
P2X7R mutants, EGFP was tagged to the C-terminal of Cys572
The channel properties of the C-terminal point mutants were examined in
Xenopus oocytes. The foremost observation with the oocytes
was that they did not reliably recapitulate the data obtained from
HEK-293 cells. Of the three point mutants that failed to express at the
surface and consequently failed to function as channel, only one of
these, Cys572 Cell Surface Expression and Pore Formation Can Be Regulated by a
Distal Region of the Cytosolic C-terminal--
Progressive deletion of
the P2X7R reveals three functional classes of truncation mutant. The
first class is truncated receptors retaining 10-80% of the C-terminal
tail that display normal channel function but have complete loss of
pore function. The second class is truncated receptors retaining 95%
or more of the C-terminal tail that display normal channel and pore
activity. The third class comprises truncated receptors retaining less
than 10% (between 80 and 95%) of the C-terminal domain or those that
have been truncated in the region between residues 551 and 581. Such
truncations are completely non-functional and do not display surface
expression. These data are consistent with the idea that the 551-581
region regulates cell surface expression of the full-length
pore-forming P2X7R. Furthermore, because we could not separate the cell
surface expression region from the region required for pore formation, this indicates that the structural elements required for pore formation
are closely associated with an additional trafficking domain.
Analysis of point mutations within this distal region of the cytosolic
C-terminal provides additional evidence of its involvement in cell
surface expression. Functional and receptor localization data of
Cys572 Is the Putative Surface Expression/Pore-enabling Domain Identical
to the Putative LPS Binding Domain?--
The functional role of
the C-terminal pore-enabling domain may be to promote pore formation by
ensuring an altered biogenesis pathway (as opposed to one for channel
forming P2X7 truncations) or to anchor the C-terminal tail to the
membrane by interacting with membrane phospholipids. The
551-RSYATWRFVSQDMADFAILPSCCRWKIRKEFP-582 region partially
overlaps a previously identified LPS binding domain found in the human
P2X7R and seen in the rat, 573-CRWRIRKEFPKSEGQYS-589 (residues that overlap are underlined). Denlinger and colleagues (16)
found that a peptide made with the above residues of the human P2X7R
could neutralize LPS-induced activation of intracellular kinases. This
region may also promote heteromeric interactions between the P2X7R and
other membrane proteins (17). Recently, the interaction of the P2X7R
(through its C-terminal tail) with epithelial membrane proteins was
found to mediate P2X7-induced cell blebbing (18). Perhaps the presence
of the pore-enabling domain within the C terminus tail augments the
P2X7 receptor's pore-forming ability as well as the cytolytic
consequences, through the interaction with protein partners.
The Surface Expression/Pore-enabling Domain of the P2X7 Receptor Is
Not Shared with Other P2X Receptors--
Pore formation has been found
to be a property shared by a number of P2X family members with both the
P2X2R and P2X4R reported to form pores (24, 25). The fact that these
receptors are able to form pores but do not contain the P2X7
pore-enabling domain (residues 551-581) indicates that a separate P2X
pore-forming domain may exist. Khakh and Lester (7) proposed that the
change between channel and pore states was achieved through a
conformational change within the selectivity filter and have recently
reported that the permeation of the I2 state of the P2X2R
is modulated by specific residues (432 and 444) within the C-terminal
tail (26). The truncated P2X2aR-(1-403) was reported to be
permeable to large cations (positively charged
N-methyl-D-glucamine) (25), which is in
contrast to the minimum length requirement of 582 residues for the
P2X7R to form pores seen in the present study.
Little is known about additional regions of the P2X7R that modulate
surface expression. The region preceding the second transmembrane domain appears to determine surface expression of the P2X2R,
because it regulates subunit interactions and receptor assembly (27). A
splice variant of the P2X1R (residues 175-201 are absent) does not
display cell surface expression, yet will co-assemble with wild type
P2X1 subunits (28). Also, the trafficking of the human P2X1R to
the cell membrane is reduced if the disulfide bridges between the
cysteine residues (in the extracellular loop) are disrupted,
specifically within Cys251-Cys270 and
Cys117-Cys165 (13).
A Working Model of the Distal C-terminal Region--
We have
generated a simple working model (Fig. 6)
that encapsulates much of the data presented in this study and permits
further analysis of the role of the distal C-terminal region in
biogenesis or in the regulation of the interaction between the P2X7R
and protein partners. This model describes a pore-enabling region situated between the residues 551-582 that also contains two sections. The first section (retention region) inhibits receptor trafficking, whereas the second section overrides the first, enabling surface expression of the P2X7R. If the integrity of this region is disrupted by a truncation or point mutation, then disruption of the associated regions exposes the retention sequence thereby blocking cell surface expression. The retention of the P2X7R may occur because a protein partner can no longer interact with the P2X7R or the P2X7R precedes into an altered biogenic pathway. A preliminary Prosite (available at
www.expasy.ch/prosite) analysis of the C-terminal tail of the rat P2X7R
identified potential phosphorylation sites for protein kinase A
(Arg557-FVSQD-Met563) and protein
kinase C (Ser552-YATWR-Phe558)
within this region. It is tempting to suggest that a
phosphorylation-dependent mechanism may govern the
regulation of these two regions, thereby providing an additional
pathway for controlling surface expression.
Physiological and Clinical Significance of P2X7 Receptor Cell
Surface Expression--
The P2X7R on hemopoietic cells is now known to
play a role in host defenses against certain infective diseases. The
function of monocyte P2X7R is increased by 5-fold on differentiation of these cells to macrophages (29) and activation of macrophage P2X7R by
extracellular ATP leads to killing of intracellular Mycobacteria Tuberculosis by these cells (30). The polymorphic P2X7R E496A receptor traffics to the cell surface but is non-functional when expressed at low surface density (29). The pathological consequence of
the Glu496
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells. The 1-Pro582Gly construct was created as above, but a
point mutation was introduced at the appropriate position within the
reverse primer (reverse: 5'-GCG GAA TTC TCA GCC
GAA CTC CTT CCG GAT CTT CC).
Gly, Glu580
Gly, and Phe581
Gly, and the wild type rat P2X7R
were subcloned into the pEGFP-N1 vector
(Clontech, Palo Alto, CA) to create four
EGFP fusion proteins. A single set of forward and reverse primers was
used to subclone all mutants and the wild type rat P2X7R into the
pEGFP-N1 vector, with an XhoI and a
KpnI site introduced at the 5' and 3' ends, and the stop
codon was deleted. All constructs were confirmed by sequencing (Australian Genome Research Facility, St. Lucia, Queensland, Australia).
Gly-EGFP, Glu580
Gly-EGFP, or
Phe581
Gly-EGFP using the Effectene transfection
reagents as described previously (9). HEK-293 cells were also
transfected with the truncated P2X7 receptors and C-terminal mutants in
preparation for calcium influx studies.
70mV with an Axoclamp 2B amplifier (Axon Instruments, Union City,
CA). Oocytes were perfused (at 2 ml/min) with a low divalent ND96
solution (in millimolar; NaCl 96, KCl 2, BaCl2 0.1, HEPES 5, pH 7.5) administered with a gravity-fed manifold perfusion system. 1 mM ATP was added for a period of 20 s and washed, and the inward current trace was monitored until full recovery was observed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Membrane topology of the rat P2X7R depicting
sites where truncations and a single point mutation were made. A
total of sixteen truncations were engineered. The number
beside the arrow designates the position of the final
amino acid for each truncation construct. Note that in one case the
terminal amino acid was also substituted (Pro582 Gly).
Seven amino acid residues within the 540 and 595 domain of the rat
P2X7R C-terminal tail were targeted for mutagenesis. A total of seven
site-directed glycine mutants were made of the full-length P2X7R;
targeted residues are shown in boldface and are
underlined.
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Fig. 2.
Pore and channel activity measured in HEK-293
cells transfected with truncated P2X7 receptors. A, a
typical time-course curve seen for cumulative ethidium ion uptake under
low divalent conditions, for HEK-293 cells transiently transfected with
the wild type rat P2X7R. Note the lack of ethidium ion uptake for
HEK-293 cells transfected with vector alone. BzATP (100 µM) was present throughout the entire experiment
(arrow indicates the time of agonist application). The
relative fluorescence intensity (RFI) is a measure of
fluorescence intensity under a fixed set of illumination and detection
parameters. B, the cumulative level of ethidium ion uptake
calculated for each truncated receptor after 30 min in the continued
presence of BzATP (100 µM) under the same conditions as
A. The number of wells examined for each truncation is shown
in parentheses. *, p < 0.05 (one-way
ANOVA), significantly different from the wild type rat P2X7R.
C, in HEK-293 cells, the time course of fluo-4 fluorescence
(recorded every 3 s for a total of 5 min) in response to BzATP (50 µM) in high divalent HEPES buffer. The wild type rat
P2X7R gave a robust and long-lived response, whereas a transient
response, due to the presence of endogenous P2Y receptors, was observed
in HEK-293 cells transfected with the vector alone. The
arrow indicates the time at which BzATP (50 µM) was applied. D, the integral of the fluo-4
fluorescence (or channel (Ca2+) activity index) was
measured for a 5-min period following BzATP (50 µM) in
each cell (total number is given in parentheses), then
averaged. Experiments were performed under identical ionic conditions
to those in Fig. 2C. *, p < 0.05 (one-way
ANOVA), significantly different from the wild type rat P2X7R; #,
p < 0.05 (one-way ANOVA), significantly different from
pcDNA3.1(+)-transfected cells. Normalized values for the surface
expression of each truncated receptor are presented in
italics.
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Fig. 3.
Comparison of ATP-induced current recordings
of wild type rat P2X7R and truncated P2X7 receptors expressed in
Xenopus oocytes. A, application
of 1 mM ATP for 20 s (as indicated by downward
arrows above each trace) to oocytes expressing the
truncated P2X7 receptors, induced an inward current that recovered
8-10 min after agonist application. All experiments were performed in
a low divalent ND96 solution. B, peak current was measured
isochronically (I1 ( ), 20 s post 1 mM
ATP addition; I2 (
), 4 min post 1 mM ATP
addition) for each truncated P2X7R and the wild type rat P2X7R. The
number of oocytes is shown in parentheses. # and *,
p < 0.05 (one-way ANOVA) significantly different from
the I1 or I2 phase of the wild type rat P2X7R.
A summary of the functional properties of the truncated P2X7 receptors
heterologously expressed in HEK-293 cells compared with those measured
in Xenopus oocytes. A plus or minus
sign has been assigned to the functional data obtained from
HEK-293 cells. A plus sign denotes responses that were
statistically indistinguishable from wild type control; a minus
sign denotes those that were significantly different from wild
type control. Truncated receptors that displayed 16% or greater P2X7R
surface expression (compared with the wild type rat P2X7R) were
assigned a plus sign; any receptor that was less then 16%
were denoted with a minus sign.
Gly, Arg574
Gly, and Phe581
Gly did
not accumulate ethidium ions in response to BzATP (Fig. 4A). In contrast,
Cys548
Gly, Lys576
Gly,
Glu580
Gly, and Pro582
Gly, all
displayed a BzATP-elicited ethidium ion uptake identical to that of
wild type P2X7R (Fig. 4A).
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Fig. 4.
Glycine-scanning mutagenesis reveals residues
vital for P2X7R function and cell surface expression.
A, pore formation was determined in HEK-293 cells
transfected with the C-terminal mutants by measuring ethidium ion
uptake 30 min after the addition of BzATP (100 µM). All
experiments were performed in identical conditions to those used in
Fig. 2A. The number of wells examined for each mutant is
given in parentheses. *, p < 0.05 (one-way
ANOVA), significantly different from wild type rat P2X7R. B,
HEK-293 cells transfected with the mutant receptors were loaded with
fluo-4 AM and fluorescence intensity monitored during BzATP (50 µM) application. All experiments were performed in
identical conditions to those used in Fig. 2C. The
integrated fluo-4 intensity (or channel activity index) over a 5-min
period is shown. The number of cells examined for each mutant is given
in parentheses. *, p < 0.05 (one-way
ANOVA), significantly different from the wild type rat P2X7R.
Normalized values for the surface expression of each mutant receptor
are presented in italics. C, confocal imaging of
EGFP-tagged receptors reveals that non-functional C-terminal mutants
fail to localize to the plasma membrane. Wild type P2X7R-EGFP and
P2X7R(E580G)-EGFP display distinct plasmalemma localization in either
HEK-293 (i) or COS-7 (ii) cells. In contrast, the
P2X7R(C572G)-EGFP and P2X7R(F581G)-EGFP mutants are retained in the
cytosol or endoplasmic reticulum. The scale bar in the
upper panels (HEK-293) represents 10 µm and 5 µm in the
lower panels (COS-7).
Gly,
Arg574
Gly, and Phe581
Gly mutants that
failed to accumulate ethidium ions in response to 100 µM
BzATP also failed to show BzATP-activated calcium influx (Fig.
4B). As we expected, mutants that displayed robust ethidium accumulation displayed wild type levels of calcium influx (Fig. 4B).
Gly, Glu580
Gly, and Phe581
Gly.
Fusion proteins were expressed in both HEK-293 and COS-7 cells.
Confocal microscopy revealed that the GFP fluorescence of both
Cys572
Gly-EGFP and Phe581
Gly-EGFP
constructs was localized to the endoplasmic reticulum and cytosol (Fig.
4C). However, the wild type rat P2X7R-EGFP and Glu580
Gly-EGFP proteins had a distinct plasmalemma
distribution with identical expression patterns in both HEK-293 and
COS-7 cells (Fig. 4C).
Gly, had a marked functional deficit when
expressed in Xenopus oocytes (Fig.
5, A and B). In
this case the application of 1 mM ATP to oocytes injected
with the Cys572
Gly mutant yielded a significantly
reduced I1 current and an absence of I2 current
(Fig. 5B). There was a less marked functional deficit seen
in the, Phe581
Gly mutant, that displayed an attenuated
I1 current with a wild type I2 current (Fig.
5B). The ability of expression in Xenopus oocytes
to rescue function is likely to represent differences in protein
trafficking, post-translational modification, or presence of
interacting proteins between mammalian and amphibian expression systems.
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Fig. 5.
Comparison of ATP-induced current recordings
of wild type rat P2X7R and C-terminal point mutants expressed in
Xenopus oocytes. A, application of 1 mM ATP for 20 s (indicated with a downward arrow
above each trace) to oocytes expressing the C-terminal
point mutations induced an inward current that recovered 8-10 min
after application. All experiments were performed in a low divalent
ND96 solution. B, current was measured isochronically
(I1 ( ), 20 s post 1 mM ATP addition;
I2 (
), 4 min post 1 mM ATP addition) and
averaged across experiments. The number of oocytes used for each
average is shown in parentheses. #, p < 0.05 (one-way ANOVA) significantly different to the I1 of
the wild type rat P2X7R.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gly, Arg574
Gly or
Phe581
Gly P2X7R mutants that showed complete loss of
function and a complete lack of cell surface expression indicate that
these residues have key roles in determining receptor localization of pore-forming receptors. Although the C-terminal
"surface-expression/pore-enabling" domain of the P2X7R is essential
for pore formation, its presence is not necessary for channel function.
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Fig. 6.
Diagram depicting a possible model for
C-terminal function domains of the P2X7 receptor. The retention
region (R) is shown together with the R
inhibitory region (R-IR). The inset shows the
normal state of the receptor that would be necessary for cell surface
expression. The model predicts that disruption of this
interaction would result in impaired cell surface expression. The
pore-enabling domain is also shown and likely interacts with the
retention domains in some fashion to promote pore formation. The
dashed line marks the putative LPS-binding domain
(16).
Ala polymorphism is the lack of
agonist-mediated P2X7R apoptosis in B-lymphocytes in patients with
chronic B-lymphocytic leukemia (19), and patients' susceptibility to
tuberculosis is the subject of current investigation. Regulation of
P2X7R cell surface expression may represent an important pathway for
modulating receptor function in normal and pathological states.
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FOOTNOTES |
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* This work was supported in part by National Health and Medical Research Council (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.
§ Supported by a scholarship from the Anti Cancer Council of Victoria (Australia).
** Supported by a National Health and Medical Research Council project grant to Michael W. Parker.
To whom correspondence should be addressed. Tel.:
61-3-8344-5833; Fax: 61-3-8344-5818; E-mail:
spetrou@unimelb.edu.au.
§§ Supported by the Cure Cancer Australia Foundation and Leukaemia Foundation of New South Wales (Australia).
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211094200
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ABBREVIATIONS |
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The abbreviations used are: P2X7R, P2X7 receptor; BzATP, 3'-o-(4-benzoyl)benzoyl ATP; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; ANOVA, analysis of variance; FACS, fluorescence-activated cell sorting; LPS, lipopolysaccharide.
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REFERENCES |
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