1 The Laboratory of Biophysics and Molecular Physiology and 2 The Laboratory of Confocal and Fluorescence Imaging, Department of Physiology, The University of Melbourne, Victoria 3010, Australia
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ABSTRACT |
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The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.
pore-forming protein; cytolytic; HEK-293; P2Z; enhanced green fluorescent protein; fusion protein
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INTRODUCTION |
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THE P2X PURINERGIC RECEPTOR family contains seven members (P2X1 through P2X7). All members possess two transmembrane domains connected by a large extracellular loop and form ATP-gated ion channels (see Ref. 26 for review). The P2X7 receptor, first cloned from the rat brain (32), encodes a 595-amino acid protein that is structurally divergent from the other P2X subtypes, because it has a long (~200 amino acids) intracellular carboxy tail. This receptor is predominantly expressed in immune cells including macrophages, lymphocytes, and mast cells (5), although its expression is not restricted to cells of hemopoietic origin.
The activated P2X7 receptor has been shown to induce cell death either by an apoptotic (20) or necrotic pathway (34). It is this death-related function that implicates P2X7 receptor involvement in a number of pathological states including cancer (8), neurodegenerative diseases (12), and ischemic penumbras (25). Curiously, a benefit of this P2X7-induced cell death is the destruction of Mycobacterium tuberculosis residing within infected macrophages (7, 19).
A unique property of the P2X7 receptor is that it exhibits two agonist-activated conductance states. Upon activation with ATP or the potent agonist benzoylbenzoyl-ATP (BzATP), the receptor becomes permeant to small cations such as Na+, K+, and Ca2+ (34). Repeated or prolonged application of either agonist causes dilation of the pore, such that it is permeable to larger cations like ethidium ion or N-methyl-D-glucamine (11, 13, 27, 35). The mechanism underlying this pore dilation is unknown. Studies by Cockcroft and Gomperts (4) suggested that this dynamic selectivity is the result of successive oligomerization of monomeric subunits, resulting in a dilated pore permeable to larger cations. This is somewhat analogous to the dynamic oligomerization seen in a number of toxins (24, 31). An alternative mechanism is that a simple conformational change in the channel's selectivity filter is responsible for pore dilation or dynamic selectivity (14).
The capability of the P2X7 receptor to form pores (in endogenous cells) has been correlated with its relative surface expression, such that pore formation may require a threshold number of agonist-bound P2X7 receptor subunits (9). If this is indeed the case, then an increase in receptor density may encourage oligomerization events and subsequent pore formation.
Fluorescent tags such as green fluorescent proteins (GFPs; or variants of GFP) or fluorescently labeled antibodies have been used to examine receptor localization and receptor interactions with other proteins. Macroscopic changes such as alterations in receptor density, including receptor translocation to the cell membrane or real time receptor clustering, have been studied with tagged biomolecules (3, 36). In particular, GFP has been tagged to the carboxy termini of both the P2X1 and P2X2 receptors (6, 15) and expressed in neuronal cells. Interestingly, the P2X1-GFP construct was found to form "synaptic-size" clusters (1 µm) in the plasma membrane that were internalized on agonist addition (6). Examination of the P2X2-GFP chimera revealed that agonist addition induced varicose "hot spot" formation (1-2 µm) but not receptor internalization (15).
In the present study a similar approach was exploited to determine whether real time macroscopic clustering coincides or precedes pore formation of the rat P2X7 receptor. We define these clustering events as the appearance of localized areas of bright GFP fluorescence (hot spots) present on the plasmalemma that range from 1 to 2 µm in size. The formation of clusters may induce oligomerization of the P2X7 receptor, by increasing the probability of subunits to interact with one another because of an increase in receptor density. For this purpose, GFP-tagged P2X7 receptors were generated and their functional properties characterized. Pore formation was determined by ethidium ion uptake in both HEK-293 and COS-7 cells, and channel conductance was measured in Xenopus oocytes with the two-electrode voltage-clamp method. We examined whether GFP-tagged P2X7 receptors formed hot spots in HEK-293 and COS-7 cells, by performing a three-dimensional analysis of images taken with a confocal laser scanning microscope.
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EXPERIMENTAL PROCEDURES |
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Materials. pEGFP-N1 and pEGFP-C1 vectors were purchased from Clontech (California) and the pCI vector from Promega (Sydney, Australia). The Ambion T7 mMessage mMachine mRNA kit was sourced from Gene Works (Adelaide, Australia), and the Chroma Spin-100 columns were from Progen Industries (Brisbane, Australia). DMEM, penicillin-streptomycin, T4 DNA ligase, trypsin-EDTA, and ethidium bromide were purchased from Life Technologies (Melbourne, Australia). ATP, BzATP, poly-L-lysine, and trypan blue were all purchased from Sigma-Aldrich (Melbourne, Australia). Fetal bovine serum (FBS) and the Effectene transfection reagent kit were purchased from Commonwealth Serum Laboratories (Melbourne, Australia) and Qiagen (Melbourne, Australia), respectively.
Molecular biology.
Enhanced GFP (EGFP)-tagged P2X7 receptors were created by subcloning
the rat P2X7 receptor (rP2X7R) into the multiple cloning site of the
pEGFP-N1 or the pEGFP-C1 vector. To generate
the P2X7-EGFP construct, the rP2X7R was PCR amplified to introduce
XhoI and KpnI restriction sites at the 5' and 3'
ends, respectively, and to remove the stop codon. EGFP-P2X7 fusion was
created by PCR amplification of rP2X7R to introduce the XhoI
restriction site and delete the ATG start codon at the 5' end, with a
HindIII site at the 3' end of the rP2X7R. The rat
N-methyl-D-aspartate (NMDA)-1A-EGFP construct was created using PCR amplification, with a
HindIII site at the 5' end and a BamHI site at
the 3' end. All PCR products and the vectors were digested with
appropriate restriction enzymes and ligated overnight using T4 DNA
ligase. Ligated products were transformed into competent DH5 cells,
and individual colonies were screened using standard restriction
analysis procedures.
Cell culture. HEK-293 and COS-7 cells were grown at 37°C with 5% CO2 in DMEM, supplemented with 10% FBS and penicillin-streptomycin (100 U/ml). For imaging studies, the cells were trypsinized with trypsin-EDTA and seeded onto poly-L-lysine-treated coverslips. After 24 h, cells were transfected with P2X7-EGFP, EGFP-P2X7, or wild-type rP2X7R with Effectene reagent. To reduce cytotoxicity, HEK-293 cells were transfected for 5 h, washed, and then further incubated in complete DMEM medium. COS-7 cells were transfected overnight to increase transfection efficiency. Transfection efficiency was ~65-75% in HEK-293 cells but only 40% in COS-7 cells.
Confocal microscopy: receptor localization and pore formation. Transfected cells were viewed on a Bio-Rad MRC-1024 confocal microscope using the 488-nm line of the Argon laser. Fluorescent signals from EGFP and ethidium ion were viewed simultaneously in separate detector channels and sampled every 150 s. Time course data were collected using the Laser Sharp "Timecourse" software module (version 3.1). Recordings were made in a low divalent HEPES buffer (in mM: 147 NaCl, 2 KCl, 10 HEPES, 10 glucose, and 0.1 CaCl2, pH 7.4). ATP and BzATP were prepared in HEPES buffer (low divalent) containing ethidium bromide (10 µg/ml). Pore formation experiments were performed 48 h after transfection when plasmalemma localization in both HEK-293 and COS-7 cells was most evident. Ethidium ion uptake experiments were carried out at either room temperature (~24°C) or at 37°C. Experiments conducted at 37°C were performed using a water-jacketed immersion lens, and solutions were heated to the required temperature before experimentation. Ethidium ion fluorescence was measured in the nucleus of individual cells 10 min after agonist application. For each construct (at each agonist dose) four separate coverslips were analyzed, and on average 10 cells in a field were measured.
Three-dimensional images of transfected cells were obtained with an Olympus Fluoview laser scanning confocal microscope and software (FV500). Cells were imaged (Olympus ×40 water immersion lens, numerical aperture 0.9) for green fluorescence by excitation with the 488-nm line from an argon laser. A series of 30-40 images (Z-spacing 0.6 µm) was collected from a single cell, before and at specified time points (detailed in text) after agonist application. The two-dimensional tomographic images were reconstructed using the three-dimensional analysis module within the Fluoview software. Separate analysis of fluorescent beads (Molecular Probes, Eugene, Oregon) indicated that the optical system would be expected to resolve clusters >0.6 µm in diameter.Trypan blue exclusion experiments. HEK-293 cells transfected with wild-type rP2X7R, P2X7-EGFP, or EGFP-P2X7 were tested for trypan blue exclusion on addition of BzATP (10 and 100 µM) at room temperature (~24°C). Uptake of ethidium ion and the visualization of trypan blue (0.4%) were monitored concurrently over a period of 2 h, using an epifluorescence microscope (Nikon Diaphot 300).
Expression and channel properties of EGFP-tagged rP2X7Rs.
Oocytes from adult female Xenopus laevis (Disa Exporters,
Somerset West Cape, South Africa) were surgically removed and prepared as outlined previously (29). Stage 5 or 6 oocytes were
injected with cRNA encoding wild-type rP2X7R, P2X7-EGFP, or EGFP-P2X7
and were stored at 18°C for 48 h before experimentation. For two
electrode voltage-clamp recordings, oocytes were impaled with glass
electrodes containing 3 M KCl and held at a command potential of 70
mV using an Axoclamp 2B amplifier. Oocytes were perfused (at 2 ml/min) with a low divalent ND96 solution (in mM: 96 NaCl, 2 KCl, 0.1 BaCl2, and 5 HEPES, pH 7.5) using a gravity-fed manifold
perfusion system. Different concentrations of ATP (0.33, 1, 3.3, 10, 33, 100, or 333 µM, or 1 mM) were applied to each oocyte until a peak current was achieved. Additional doses of ATP were applied after a
20-min washout period. EC50 values were determined from the responses of each oocyte.
Data analysis. Electrophysiological analysis of two-electrode voltage-clamp results was performed using CLAMPFIT (Axon Instruments) or ORIGIN 6.0 (Microal Software). Data in the text and figures are shown as means ± SE, and statistical analysis was performed using a Student's t-test. Data from ethidium ion uptake experiments were analyzed as described in the text and are presented as means ± SE. Statistical analysis was performed using a one-way ANOVA (Tukey's pairwise comparison).
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RESULTS |
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The P2X7-EGFP and EGFP-P2X7 constructs were designed to examine whether pore formation precedes or coincides with a change in receptor distribution, typically referred to as formation of receptor hot spots (15), which would be detectable with standard confocal microscopy. Before these constructs could be used for this purpose, it was important to characterize both the channel and pore-forming properties of the GFP-tagged receptors and compare them with properties of the wild-type rP2X7R. Channel properties were examined by two-electrode voltage-clamp analysis of Xenopus oocytes, and pore formation was studied by ethidium ion uptake in transfected HEK-293 or COS-7 cells.
Expression and channel properties of P2X7-EGFP, EGFP-P2X7, and
wild-type rP2X7Rs.
Application of 1 mM ATP to oocytes injected with either P2X7-EGFP,
EGFP-P2X7, or wild-type rP2X7R cRNA induced a rapid inward current that
recovered over a 5- to 6-min period, as illustrated in Fig.
1. To test the ATP sensitivity
of fusion proteins, solutions with ATP in the concentration range from
0.33 µM to 1 mM were applied, and maximum peak currents were recorded
and normalized to generate ATP dose-response curves. These curves
revealed a reduced ATP sensitivity of the P2X7-EGFP receptor, compared
with both EGFP-P2X7 and wild-type rP2X7R (Fig.
2). Altered receptor characteristics of
P2X7-EGFP were further reflected in differences in the EC50
and the Hill coefficient. The EC50 value of wild-type rP2X7R was 4.9 ± 0.5 µM, compared with 6.0 ± 0.6 µM for
EGFP-P2X7 and 25.5 ± 6.4 µM for P2X7-EGFP. The Hill coefficient
calculated for P2X7-EGFP was 1.5 ± 0.2, which was significantly
lower than that calculated for both wild-type rP2X7R (2.2 ± 0.2)
and EGFP-P2X7 (2.5 ± 0.5). Interestingly, there was no
significant difference in the maximum peak current between wild-type
rP2X7R (2.84 ± 0.35 µA), P2X7-EGFP (2.76 ± 0.50 µA),
and EGFP-P2X7 (2.72 ± 0.26 µA). Oocytes injected with P2X7-EGFP
or EGFP-P2X7 cRNA exhibited green fluorescence when examined by
confocal fluorescence microscopy. The application of BzATP to injected
oocytes induced inward currents that generally took >30 min to recover
(with low divalent conditions); hence, full BzATP dose-response curves
were difficult to obtain, and ATP was used (29).
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Ethidium ion uptake in HEK-293 or COS-7 cells.
Pore formation was examined by a standard agonist-activated ethidium
ion uptake procedure (13). HEK-293 cells transfected with
either P2X7-EGFP, EGFP-P2X7, or wild-type rP2X7R accumulated ethidium
ion on agonist application (10 and 100 µM BzATP; Fig. 3A). Ethidium ion fluorescence
was augmented at 37°C, particularly in HEK-293 cells transfected with
P2X7-EGFP. Simultaneous measurement of EGFP and ethidium ion
fluorescence revealed that cells exhibiting EGFP fluorescence were also
positive for ethidium ion fluorescence after prolonged application of
BzATP indicative of pore formation (Fig. 3B), and within 30 min all EGFP-positive cells displayed an intense ethidium ion
fluorescence in the nucleus. The bright ethidium ion fluorescence was
not the result of membrane damage, because under the same conditions
cells excluded trypan blue.
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Do receptor hot spots or increases in receptor density correlate
with pore formation?
Cellular localization of tagged rP2X7Rs was examined with confocal
microscopy. Twenty-four hours after transfection of HEK-293 cells, the
distribution of EGFP fluorescence was similar to that of the
endoplasmic reticulum revealed by Di-O6 staining (data not shown).
After 48 h, the fluorescence of both amino- and
carboxy-terminal-tagged rP2X7Rs was apparent in the plasmalemma with
some residual cytosolic signal (Fig. 5,
A and B). In COS-7 cells, P2X7-EGFP was
effectively targeted to the plasmalemma (Fig. 5C), whereas
EGFP-P2X7 showed a more diffuse cytosolic localization (Fig.
5D). A number of multinucleated cells were present in
transfected HEK-293 cells (expressing tagged or wild-type
rP2X7Rs), as previously reported (2), but this phenomenon was never observed in transfected COS-7 cells.
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DISCUSSION |
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The mechanism by which agonists such as ATP induce P2X7 receptors to change ion size selectivity is still not known. A number of intra- and intermolecular models have been proposed, which describe the process that accounts for the change in ion selectivity of this receptor. Intramolecular models suggest that subtle structural changes, possibly in the selectivity filter itself, contribute to the dynamic change from channel to pore state (14). Intermolecular models predict that pore formation occurs by dilation of the channel, wherein successive addition of extra subunits produces an oligomeric pore capable of conducting large size (~900 Da) cations (4). This latter possibility was the focus of the present studies. If a real time oligomerization is responsible for pore dilation, then real time subunit redistribution or clustering may be visualized by imaging EGFP-tagged rP2X7Rs. Because it was not known whether EGFP tagged to either the amino or carboxy terminus would affect the functional properties of the P2X7 receptor, we examined the functional consequences of fusing EGFP to either end of the rP2X7R.
Two-electrode voltage-clamp recordings revealed that ATP-induced currents of the fusion constructs were similar to those of wild-type rP2X7R and to previously published work (29). Wild-type rP2X7R displayed higher sensitivity to ATP when expressed in oocytes (29) than in HEK-293 cells (32), although activation curves of both systems were best described by Hill coefficients of ~2 (30). P2X7-EGFP fusion was shown to have a decreased ATP sensitivity compared with both wild-type or EGFP-P2X7 receptors. The decreased ATP sensitivity of P2X7-EGFP indicates that EGFP tagged to the carboxy terminus disrupts either the binding of ATP to the receptor or alters transduction subsequent to the binding event. A disruption of ATP binding is unlikely, since ATP binding occurs within the extracellular loop (10) and both the carboxy and amino terminals are cytosolic. This result contributes to the growing evidence of the involvement of the carboxy terminal tail in regulating the function of the P2X7 receptor.
The absolute requirement of the full-length carboxy-terminal tail for P2X7 pore formation has been previously established (32). Until recently, little was known about the involvement of the carboxy-terminal tail with respect to the channel response. Klapperstuck and colleagues (16) observed in Xenopus oocytes (expressing the human P2X7 receptor) that the carboxy-terminal tail is involved in the regulation of ATP-induced (channel) currents, as the truncated receptor (human P2X7 1-418) yielded an altered response that differed from that of the wild-type receptor. Unlike other members of the P2X family (P2X1-4), where a string of charged residues within the carboxy-terminal tail has been found to alter channel kinetics (17), the carboxy-terminal tail of the P2X7 receptor appears to be more complex, with the function of crucial regions largely unresolved.
Previous studies have also shown that ion channels tagged with GFP exhibit an altered response. In particular, Khakh and colleagues (15) revealed a rightward shift in the ATP dose-response curve of the P2X2-GFP construct when expressed in Xenopus oocytes, although there was no change in the Hill coefficient. GFP fused to the amino terminus altered the kinetics of K+ channels, although GFP addition to the carboxy terminus was not examined (18, 23). The present study highlights the importance of functional testing of both amino- and carboxy-terminal fusion constructs before other applications.
Pore formation was examined by ethidium dye uptake measurements in the continued presence of BzATP. Both amino- and carboxy-terminal tagged rP2X7Rs were permeable to ethidium ion, with the amount of ethidium ion uptake (10 min post-BzATP addition) comparable to that of wild-type rP2X7R. The observed difference in ethidium ion uptake between HEK-293 and COS-7 cells suggests that rP2X7R function may be regulated by local factors such as lipid environment or involvement of a cell-specific protein partner. In COS-7 cells ethidium ion uptake was augmented by increasing the temperature from ~24°C to 37°C, in agreement with earlier work by Cario-Toumaniantz and colleagues (1).
The involvement of a protein partner has been previously postulated as an explanation for the variant ion size selection of the P2X7 receptor, between cells and species. As yet little is known about the protein-protein interactions of the P2X7 receptor. Postulated protein partners might play the role of pore-enabling or pore-inhibiting factor. The P2X7 receptor is known to form homooligomers, but heteroligomers with other members of the P2X family are not apparent (33). Interestingly, Parker (28) observed that cytoskeletal actin interacts with and regulates the kinetics of the P2X1 receptor. Likewise McKenzie and Surprenant (21) observed that F actin is involved in the blebbing induced by P2X7 activation. Further investigation into the regulation of pore formation by P2X7 protein partners or the interaction of the receptor with cytoskeletal proteins is warranted.
Confocal imaging studies showed plasmalemma localization of rP2X7R in HEK-293 and COS-7 cells. However, the location of the EGFP does affect the expression pattern. EGFP-P2X7 expressed in HEK-293 cells could be localized to the plasmalemma, although this was not the case when this construct was expressed in COS-7 cells. This difference is intriguing and may point to the involvement of protein partners that are required for trafficking and expressed at varying levels in different cell types.
An increase in membrane density of rP2X7R has been proposed to augment pore formation (9). It is peculiar that COS-7 cells expressing P2X7-EGFP exhibit intense plasmalemma fluorescence but only a mild pore-forming phenotype, suggesting that there is no simple relationship between receptor density and propensity to form pores.
Recent studies examining the localization of the P2X1-GFP fusion protein demonstrated synaptic size clusters in the plasma membrane of HEK-293 cells and dissociated neurons (6), although these receptor clusters were present before agonist application. Agonist-induced changes in GFP-tagged rat P2X2 receptors expressed in neuronal cells could be readily observed in dendrites, demonstrating that real time receptor redistribution is a physiologically significant response to receptor activation (15). In contrast, this study reveals that P2X7 pore formation does not involve either macroscopic receptor density changes or receptor clustering.
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NOTE ADDED IN PROOF |
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A proteomic analysis of P2X7 protein partners has been recently published: Kim M, Jiang LH, Wilson HL, North RA, and Surprenant A. Proteomic and functional evidence for a P2X7 receptor signalling complex. EMBO J 20: 6347-6358, 2001.
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ACKNOWLEDGEMENTS |
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We thank both reviewers for their helpful and insightful comments.
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FOOTNOTES |
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This project was supported by the Anti Cancer Council of Victoria (Australia) and the William Buckland Foundation (ANZ Trustees, Australia).
Address for reprint requests and other correspondence: S. Petrou, The Laboratory of Biophysics and Molecular Physiology, The Univ. of Melbourne, Victoria 3010, Australia (E-mail: spetrou{at}unimelb.edu.au).
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.
First published February 13, 2002;10.1152/ajpcell.00456.2001
Received 24 September 2001; accepted in final form 12 February 2002.
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