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Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada (R.R., Y.C., D.B., A.S., P.S.); Unité Mixte de Recherche 5543, Centre National de la Recherche Scientifique, Université Victor Segalen Bordeaux 2, Bordeaux, France (E.B.-G.); and AstraZeneca R&D Charnwood, Loughborough, England (D.H.)
Received July 1, 2003; accepted November 21, 2003
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Abstract |
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). The P2X7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage, including microglial and dendritic cells (Collo et al., 1997). One distinct structural feature of P2X7 receptor-channels, a large intracellular carboxyl-terminal domain (239 amino acids in mammalian subunits), is involved in the progressive formation of large pores permeable to molecules up to 900 Da (Surprenant et al., 1996; Virginio et al., 1999b) as well as in the efficient release of the proinflammatory cytokine interleukin 1-
, as demonstrated in a knock-out model (Solle et al., 2001). In macrophage-related cells and glia, ATP-induced P2X7 activity has been reported to be linked to a number of cellular events including membrane blebbing and microvesiculation (Virginio et al., 1999a; MacKenzie et al., 2001), cell fusion (Chiozzi et al., 1997), activation of nuclear factor-
B and nuclear factor of activated T cells (Ferrari et al., 1997, 1999), activation of p38 mitogen-activated protein kinase (Neary et al., 2003), activation of phospholipase D (Dubyak and el-Moatassim, 1993; el-Moatassim and Dubyak, 1993), killing of intracellular mycobacteria (Lammas et al., 1997; Smith et al., 2001), and cell death (Di Virgilio et al., 1998). Although it is not known whether these cellular events depend on specific functional properties of the P2X7 subtype, they underline the significant role played by P2X7 receptors in the regulation of immune response in peripheral tissues as well as in the central nervous system. Despite the potential physiological and therapeutic importance of P2X7 receptors, their pharmacology remains limited. The agonist 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) is more potent than ATP at activating rodent and human P2X7 receptors, and this marked difference in sensitivity has been naturally used as an index of the presence of native P2X7 receptors. However, their identification cannot be based solely on this differential sensitivity because BzATP has been reported to be an agonist for other P2X receptor subtypes (Bianchi et al., 1999; Boué-Grabot et al., 2000a). Irreversible blockade of P2X responses by long incubations with oxidized ATP was also considered a solid proof of the involvement of native P2X7 receptors; however, oxidized ATP has subsequently been shown to block P2X1 and P2X2 receptors (Evans et al., 1995). A monoclonal antibody directed against an extracellular epitope of P2X7 subunit has antagonistic properties, but its use is limited to human P2X7 (Buell et al., 1998). In search of better antagonists, the dye Brilliant Blue G has been identified as a potent blocker of rat P2X7 in the 10 to 100 nM range, but it blocks other subtypes in the micromolar range (Jiang et al., 2000a) and its actions on metabotropic ATP receptors have not been tested. The organic cation calmidazolium, a calmodulin antagonist and a blocker of cyclic nucleotide-gated channels (Virginio et al., 1997), is effective at blocking rat and human P2X7 receptors but does not block pore formation (Chessell et al., 1998). Another P2X7 antagonist, KN-62, a known inhibitor of calcium/calmodulin-dependent protein kinase type II, has little inhibitory effect on the rat P2X7 (Humphreys et al., 1998). The lack of unambiguous and cross-species pharmacological tools for P2X7 prompted us to develop specific sequence-based antagonists. We report here the characterization of dominant-negative mutations located in the ectodomain of P2X7 subunits that are highly effective at selectively suppressing the function of rodent as well as human P2X7 receptors. Moreover, we show that these dominant-negative P2X7 subunits have the property of knocking down the activity of native P2X7 receptor channels in immune cells. |
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Cell Culture and Transfections. HEK293 cells were cultured as described previously (Le et al., 1998). HEK293 cells stably expressing the rat P2X7 subunit (HEK-P2X7), kindly provided by A. Surprenant (University of Sheffield, Sheffield, England), were cultured in Dulbecco's modified Eagle's medium and 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad,CA) containing penicillin and streptomycin supplemented with G-418 (250 µg/ml). RAW264.7 cells were kindly provided by M. Desjardins (University of Montreal, PQ, Canada) and were cultured in Dulbecco's modified Eagle's medium and 10% heat-inactivated fetal bovine serum containing 10 mM HEPES buffer (Sigma, St. Louis, MO), 5% L-glutamine (Invitrogen), penicillin, and streptomycin. Cells were transfected with Polyfect (QIAGEN, Valencia, CA) or Fugene 6 (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocols. Transfected cells were used for electrophysiological recordings 24 to 48 h after transfection. Oocytes were surgically removed from Tricaine-anesthetized female Xenopus laevis frogs and were incubated with calcium-free Barth's solution containing 1 mg/ml type I collagenase at room temperature for 2 h under vigorous agitation. Stage V and VI oocytes were then manually defolliculated before intranuclear microinjection of 1 to 10 ng of supercoiled plasmid coding for wild-type/mutant mouse P2X7, human P2X7, rat P2X7 and P2X2, or mouse 5HT3A. After injection, oocytes were incubated with Barth's solution containing 1.8 mM CaCl2 at 19°C for 24 to 72 h before electrophysiological recordings.
Electrophysiology. Two-electrode voltage-clamp recordings (VH = -60 mV) were performed using glass pipettes (1-3M
) filled with 3 M KCl solution. Oocytes were placed in a recording chamber and were perfused at a flow rate of 10 to 12 ml/min with Ringer's solution, pH 7.4, containing 115 mM NaCl, 5 mM NaOH, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES. Membrane currents (d.c., 1 kHz) were recorded using a Warner OC-725B amplifier (Warner Instrument, Hamden, CT) and digitized at 500 Hz. Drugs were dissolved in the perfusion solution and applied using a computer-driven valve system. Whole-cell patch-clamp recordings (VH of -60 mV) were performed using pipettes filled with internal solution, pH 7.2, containing 120 mM K-gluconate, 1 mM MgCl2, 4 mM NaOH, and 10 mM HEPES. Drugs were applied using a Warner SF-77B fast perfusion system at a rate of 1 ml/min. The perfusion solution, pH 7.4, comprised 145 mM NaCl, 5 mM NaOH, 3 mM KCl, 1 mM MgCl2, 0.9 mM CaCl2, and 10 mM HEPES. Membrane currents (d.c., 200 Hz) were recorded using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA) and digitized at 500 Hz. All drugs were dissolved in the perfusion solution. Current-voltage plots were obtained using a ramp protocol. All experiments were performed at room temperature.
YO-PRO-1 and Ethidium Uptake Imaging. The BzATP-dependent P2X7 mediated pore formation was assessed by imaging the uptake of YO-PRO-1 (Molecular Probes, Eugene, OR), a 629-Da propidium di-iodide dye that fluoresces when bound to nucleic acids. HEK293 cells were cotransfected with CD8 and wild-type or mutant P2X7 subunits. Twenty-four hours after transfection, dishes were rinsed twice with perfusion solution and incubated with 2 x 107 beads/ml of Dynabeads (Dynal Biotech, Lake Success, NY) for 30 min. After washing, the cells were incubated with perfusion solution containing 1 µM YO-PRO-1. BzATP (300 µM) was dissolved in low divalent solution, pH 7.4, containing 145 mM NaCl, 5 mM NaOH, 3 mM KCl, 0.09 mM CaCl2, and 10 mM HEPES. Fluorescence changes (excitation, 491 nm; emission, 509 nm) were monitored in individual cells rosetted by the beads. RAW264.7 cells were transfected with mutant subunit subcloned in pAdTrack vector containing a GFP expression cassette. After transfection (24 to 48 h), GFP-positive cells were monitored for changes in ethidium fluorescence signals (excitation, 525 nm; emission, 565 nm) in response to application of low divalent solution containing 10 µg/ml ethidium bromide in the presence or absence (control) of 3 mM ATP, pH 7.4. The fluorescence signals were corrected for baseline signal levels in control experiments. Images were captured every 30 s on a cooled charge-coupled device camera (Optikon, Kitchener, ON, Canada) and processed using Axon Imaging Workbench 2.2 software (Axon Instruments).
Immunocytochemistry and Confocal Microscopy. Transiently transfected HEK293 cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in 0.16 M phosphate buffer, pH 6.8. The cells were washed and blocked with 5% normal goat serum (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), 0.3% Triton X-100 (Fisher Scientific, Pittsburgh, PA) in phosphate-buffered saline, pH 7.4, then incubated with anti-FLAG M2 antibody (Sigma) at 4°C overnight. Antibody binding was then revealed using a Texas Red conjugated goat anti mouse (Jackson ImmunoResearch) for 60 min at room temperature. The confocal fluorescence microscopy images were acquired using an LSM 510 line scanning microscope (Zeiss, Jena, Germany).
Data Analysis. Peak currents, defined as the largest amplitudes recorded during agonist applications, were normalized to the mean of the wild-type currents under each experimental condition. Data are presented as mean ± S.E. The BzATP EC50 values were calculated by fitting the data to logistical equation using Sigmaplot software (SPSS Inc., Chicago, IL). The proportions of homomeric and heteromeric channels were calculated using binomial distribution. Assuming that heteromeric channels composed of one or more mutant subunits are silent, then the fraction of total current (I/ITotal) is obtained using the binomial equation I/ITotal = (1 - p)n where p is the proportion of mutant subunits and n is the number of subunits in the channel complex. All statistical analyses for the difference in means were carried out using Student's t tests for two unpaired groups. The analysis for the immunofluorescence intensity profiles was carried out using ImageJ software (http://rsb.info.nih.gov/ij).
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WC167-168AA Mutant Inhibits YO-PRO-1 Uptake. Activation of P2X7 receptors results in opening of nonspecific pores permeable to molecules smaller than 900 Da, such as YO-PRO-1 (Surprenant et al., 1996). We investigated whether the WC167-168AA mutant subunit can block P2X7-mediated permeabilization. As shown in Fig. 3, A and B, control cells transfected with the nonfluorescent marker CD8 alone showed no significant increase in YO-PRO-1 uptake in response to BzATP. Cells transfected with wild-type P2X7 showed a marked time-dependent increase in YO-PRO-1 uptake upon stimulation with BzATP, whereas cells cotransfected with the WC167-168AA mutant and the wild-type subunits (1:1 ratio) exhibited a significantly decreased YO-PRO-1 uptake in response to agonist (12 ± 2% of the P2X7 transfected cells, 20 min after BzATP application, n = 4).
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Impact of WC167-168AA Mutant on Receptor-Channel Properties. To determine whether the expression of the mutant causes a decrease in the agonist sensitivity, we obtained the dose-response curves for BzATP in cells transfected with P2X7 alone or in the presence of the WC167-168AA mutant (at a 2:1 ratio). As illustrated in Fig. 4A, at any given concentration, the magnitude of current in the cotransfection was smaller than the wild-type. The doseresponses exhibited a sigmoidal relationship with an EC50 of 538 ± 75 µM for the wild-type and 556 ± 31 µM for the cotransfection (n = 4) (Fig. 4B). To investigate whether the coexpression of the mutant subunit changed the ionic selectivity of P2X7 receptor-channels, we plotted the current-voltage (I-V) relationship for BzATP-evoked currents in cells transfected with wild-type subunit alone or cotransfected with the WC167-168AA subunit. In both cases, I-V relationship displayed a linear slope between -70 and +30 mV. Moreover, as shown in Fig. 4C, the reversal potential of BzATP-evoked currents in cells transfected with wild-type alone was not significantly different from that obtained in cells cotransfected with the WC167-168AA subunit (1.3 ± 2.6 mV versus -1.3 ± 1.7 mV; respectively, n = 5). These results indicate that the WC167-168AA mutant does not inhibit P2X7 receptor-channels function by reducing agonist sensitivity or by changing the relative permeability of sodium and potassium ions.
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Subcellular Localization of the WC167-168AA Mutant Subunit. To assess the subcellular distribution of the WC167-168AA subunit, we immunolocalized FLAG epitope-tagged versions of wild-type or mutant receptors in HEK293 cells. The profiles of the receptor distributions, obtained by measuring the fluorescence intensity along a line randomly drawn through the diameter of the cell, were identical for both the wild-type and mutant subunits (Fig. 5, A-C). The staining for wild-type and mutant P2X7 subunits was most intense at the edges of the cell, in contrast to the cytoplasmic distribution of EGFP in the same cell (Fig. 5D). These results show that most wild-type and mutant subunits are transported to the plasma membrane, hence indicating that the functional knock-down of P2X7 receptor is not caused by disturbed trafficking or intracellular retention.
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Potency of Inhibitory WC167-168AA and C168A Mutants. To determine the potency of the inhibitory effect of the WC167-168AA subunits on P2X7 receptors, HEK293 cells were transfected with a fixed concentration of wild-type cDNA and varying concentrations of the mutant. As illustrated in Fig. 6A, the amplitude of the current is greatly reduced (74 ± 6%) by cotransfecting wild type with mutant subunits at a ratio as low as 2:1. The amplitude of BzATP-evoked currents decreased with increasing proportions of the mutant subunit, as expected for a dominant-negative effect (Fig. 6C and Table 2).
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Next, we sought to determine the key residue involved in the inhibitory effect of the WC167-168AA subunit. We investigated the effects of single W167A and C168A mutations on the function of the P2X7 receptor. The W167A mutant was nonfunctional when expressed alone, with no obvious dominant-negative effects (data not shown). The C168A mutant, on the other hand, exhibited a substantial inhibitory effect as shown in Fig. 6B (80 ± 5% at 2:1 ratio). The dominant-negative effect increased exponentially with increasing proportions of mutant subunit (Fig. 6C and Table 2). These data indicate that the WC167-168AA and C168A mutants are highly effective inhibitory subunits for P2X7 receptors.
Knock-Down of Native P2X7 Function by Dominant-Negative Mutants. In the previous experiments, we assessed the knock-down of wild-type receptors transiently coexpressed with the mutant subunits in heterologous systems. However, in cells natively expressing P2X7 receptors, there is a pre-existing pool of surface receptors. So, as a first step to test the sensitivity of endogenous P2X7 receptors to dominant-negative blockade, we investigated the effect of the expression of the mutant subunits in HEK293 cells stably expressing P2X7 receptors (HEK-P2X7) at a high level (Surprenant et al., 1996). In HEK-P2X7 cells, transient expression of the WC167-168AA mutant subunits resulted in a significant reduction of the current responses to BzATP (15 ± 5% of control, p < 0.05, n = 3, Fig. 7A). Next, we tested the effects of expression of the dominant-negative subunits in mouse macrophage cell line RAW264.7. As shown in Fig. 7B, the dominant-negative C168A subunit significantly reduced the endogenous current responses to BzATP (31 ± 9% of control, p < 0.05, n = 3, Fig. 7B). Pore formation measured by the uptake of ethidium ions in response to application of agonist was also knocked down by transient expression of the C168A mutant subunit (25 ± 7% of control, p < 0.01, Fig. 8). These results indicate that the C168A dominant-negative subunits are able to effectively suppress both the current response and pore formation mediated by native P2X7 receptors in immune cells.
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). However, because of the presence of disulfide bridges in the ectodomain (Clyne et al., 2002; Ennion and Evans, 2002), the role of different extracellular regions in receptor function remains difficult to assess in the absence of crystallographic data. Nevertheless, we can conclude from this screening that the conserved P2X motif WC167-168 is not involved in subunit association. In our screen, the other nonfunctional subunits with mutations in the ectodomain displayed no inhibitory activity, suggesting either that they were degraded prematurely because of misfolding or that they did not associate correctly with the wild-type subunits.
Mammalian P2X7 subunits share significant homology (e.g., 80% between mouse and human), so we hypothesized that they would be able to associate into hybrid receptor-channels. We confirmed the coassembly of mouse and human P2X7 subunits by knocking down human P2X7 receptor function with mouse WC167-168AA mutant subunits (Fig. 2). The logical consequence of this compatibility of association is that dominant-negative P2X7 subunits from one species are able to suppress the function of P2X7 receptors in other species. Therefore, this genetic approach based on the expression of mutant subunits provides an advantage over knockout strategies, which are currently limited to mice. Potentially, a gene therapy approach leading to the overexpression of dominant-negative P2X7 subunits and the knockdown of human macrophage or microglial P2X7 responses could be beneficial in the control of inflammatory diseases (Cucchiarini et al., 2003).
Phylogenetically and functionally, vertebrate P2X7 subunits define a distinct gene subfamily (North, 2002). Indeed all P2X subunits have been shown to associate in heteromeric receptors (Khakh et al., 2001), except P2X7 subunits, which seem to form homomers exclusively (Torres et al., 1999). This unique property of P2X7 subunits provides a methodological advantage to selectively target the P2X7 receptors while sparing all other coexpressed P2X subtypes. By coexpressing the mutant P2X7 subunits with P2X2 subunits, we demonstrated the subtype selectivity and we confirmed the absence of heteromeric P2X2+7 ATP receptors. Likewise, the mutant P2X7 subunit did not suppress the function of unrelated homomeric 5HT3A receptor channels, chosen here as a prototypical member of the Cys-loop family of transmitter-gated channels. So, the WC167-168AA mutation conferred strong and specific dominant-negative effect to the subunit for the rodent and human P2X7 subtypes of ionotropic ATP receptors.
Several members of the P2X receptor family, including P2X7, are capable of forming large pores in response to agonist stimulation. The exact function of these pores is not known; however; given their ability to provide passage for molecules such as L-glutamate (Ballerini et al., 1996; Duan et al., 2003), they potentially play a crucial role in cell signaling and metabolism. The mechanisms linking channel activity and pore formation are not well understood. Calmidazolium has been shown to distinguish between the pore and channel states of P2X7 receptors because it can block the channel without affecting the pore formation (Virginio et al., 1997). This suggests that the pore-forming activity of P2X7 receptors may not require the opening of the channel. Given that other members of the P2X receptor family have been shown to promote pore formation (Khakh et al., 1999; Virginio et al., 1999b), an effective tool for the study of P2X7 receptors should also be able to block this function. Our YO-PRO-1 and ethidium uptake data demonstrate that the knock-down by the dominant-negative subunits is complete in that it suppresses both the channel- and the pore-forming functions of recombinant and native P2X7 receptors.
Two mechanisms by which a dominant-negative subunit is likely to exert its inhibitory effect are either by binding and sequestering the agonist away from the receptor (sink effect) or by directly associating with the receptor and blocking its function. It seems unlikely that the dominant-negative subunits described here act via a sink effect because 1) mutation of the conserved cysteines in the ectodomain of other P2X receptors has been shown to reduce the sensitivity of the receptor to ATP (Clyne et al., 2002; Ennion and Evans, 2002) and 2) the dominant-negative subunits are highly effective at blocking the wild-type receptors at low proportions. Our data show that the reduced current response has wild-type characteristics in both agonist affinity and reversal potential, indicating that channels composed of dominant-negative and wild-type subunits are silent. Hence, the dominant-negative subunits probably block the function of P2X7 receptors by association, which leads to a reduction in the number of homomeric wild-type channels.
The immunolocalization of wild-type and mutant P2X7 subunits in transfected cells and the corresponding subcellular distribution profiles show that the translocation of the dominant-negative P2X7 subunits to the plasma membrane is as efficient as the one of wild-type P2X7 subunits. We can conclude from these results that the WC167-168AA or the C168A mutant subunits do not knock-down P2X7 receptor function by intracellular retention or degradation of wildtype subunits. Heteromeric nonfunctional P2X7 receptors composed of wild-type and mutant subunits are translocated to the plasma membrane, so it confirms that the inhibition of P2X7 activity is probably caused by a decrease in the number of homomeric wild-type channels available at the cell surface.
There are 10 conserved cysteines in the ectodomain of P2X receptor family that form disulfide bonds and are important for the correct folding of the ATP binding domain (Clyne et al., 2002). Interestingly, the single mutation C168A was able to mimic the dominant-negative properties of the double mutation WC167-168AA, demonstrating the major contribution of this extracellular cysteine residue to the inhibitory effect and its importance in the normal function of P2X7 receptors. Although not silent, the rat P2X2 C164A and human P2X1 C165A mutant receptors have been shown to be deficient in trafficking to the surface (Clyne et al., 2002; Ennion and Evans, 2002). According to our immunolocalization data, substitution of Cys168 with alanine in P2X7 did not lead to disturbed trafficking of the receptors. However, the loss of a disulfide bridge could explain the inhibitory effect of the mutation through a major conformational change in the ATP binding domain or other critical domains of the receptor-channel.
In cells coexpressing the wild-type and the mutant subunits, the relative proportion of functional homomeric wildtype, silent homomeric mutant, and silent or functionally impaired heteromeric P2X7 receptors follows a statistical distribution that depends on the ratio of the subunits, on the affinity of the intersubunit associations, and on the number of subunits in the ion channel. If the affinity for intersubunit association is similar between mutant and wild-type subunits and one single subunit is necessary and sufficient to block a P2X7 channel, the binomial distribution of the different populations of receptors would translate into an exponential decrease in P2X7 current with increasing proportions of mutant subunits (Table 2).
Indeed, the experimental dose-response curves for the dominant-negative effects are clearly exponential and are compatible with a trimeric or tetrameric configuration of P2X7 receptors (Table 2). These data support a model in which one dominant-negative subunit is sufficient to knock down the function of the channel. Therefore, it provides further evidence that the inhibitory effect of these dominant-negative subunits is not merely an effect of dilution of functional subunits. P2X channels are believed to have a trimeric architecture (Nicke et al., 1998), so in native preparations, the cotranslation of only one dominant-negative mutant subunit with two wild-type endogenous subunits might generate a significant knock-down of P2X7 responses (Table 2).
When we tested the effectiveness of the dominant-negative mutant P2X7 subunits on a pre-existing pool of P2X7 receptors in a HEK293 stable cell line, we recorded a significant reduction of BzATP-induced P2X7 currents. As a proof-of-concept validating this selective dominant-negative approach, transient expression of mutant subunits in natively P2X7-expressing RAW264.7 cells also led to a significant knock-down of endogenous P2X7 receptor function. However, we noticed that in both cases, the decrease of current was lower than that observed in our cotransfection experiments. The simplest interpretation of this apparently lower inhibition in native preparations would be that the amount of cotranslation of mutant subunits with de novo wild-type P2X7 subunits is limited by the short time window of transient expression. The turnover rate of P2X7 receptors in RAW264.7 cells is not known, but half-lives as long as 10 days have been reported for other ionotropic receptors (Wang et al., 1999). Longer-term coexpression of mutant subunits with endogenous P2X7 subunits is likely to improve the effectiveness of this dominant-negative approach in macrophage-related cell types. Because these cell types are notoriously resistant to transfection (Burke et al., 2002), transgenic or viral expression of dominant-negative WC167-168AA or C168A mutant P2X7 subunits would provide more effective approaches to knock down native P2X7 responses in peripheral macrophages and in microglia.
In conclusion, dominant-negative P2X7 subunits can be used to selectively suppress the channel activity of P2X7 receptors and the associated pore formation without interfering with a wide variety of other conductances sensitive to extracellular ATP. Thus, they provide novel genetic tools to investigate the physiological and pathological role of native PX7 receptors in inflammation and in immune response.
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Acknowledgements |
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Footnotes |
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R.R. and Y.C. contributed equally to this work.
This work was presented in part at the 32nd Annual Meeting of Society for Neuroscience; 2002 Nov 2-7; Orlando, Florida.
ABBREVIATIONS: BzATP, 2',3'-O-(4-benzoyl)-benzoyl-ATP; KN-62, 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; HEK, human embryonic kidney; GFP, green fluorescent protein.
Address correspondence to: Dr. Philippe Séguéla, Montreal Neurological Institute, 3801 University Street, Suite 778, Montreal, QC, Canada, H3A 2B4. E-mail: philippe.seguela{at}mcgill.ca
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bianchi BR, Lynch KJ, Touma E, Niforatos W, Burgard EC, Alexander KM, Park HS, Yu H, Metzger R, Kowaluk E, et al. (1999) Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 376: 127-138.[CrossRef][Medline]
Boué-Grabot E, Akimenko MA, and Séguéla P (2000a) Unique functional properties of a sensory neuronal P2X ATP-gated channel from zebrafish. J Neurochem 75: 1600-1607.[CrossRef][Medline]
Boué-Grabot E, Archambault V, and Séguéla P (2000b) A protein kinase C site highly conserved in P2X subunits controls the desensitization kinetics of P2X(2) ATP-gated channels. J Biol Chem 275: 10190-5.
Buell G, Chessell IP, Michel AD, Collo G, Salazzo M, Herren S, Gretener D, Grahames C, Kaur R, Kosco-Vilbois MH, et al. (1998) Blockade of human P2X7 receptor function with a monoclonal antibody. Blood 92: 3521-3528.
Burke B, Sumner S, Maitland N, and Lewis CE (2002) Macrophages in gene therapy: cellular delivery vehicles and in vivo targets. J Leukoc Biol 72: 417-428.
Chakfe Y, Séguin R, Antel JP, Morissette C, Malo D, Henderson D, and Séguéla P (2002) ADP and AMP induce interleukin-1beta release from microglial cells through activation of ATP-primed P2X7 receptor channels. J Neurosci 22: 3061-3069.
Chessell IP, Michel AD, and Humphrey PP (1998) Effects of antagonists at the human recombinant P2X7 receptor. Br J Pharmacol 124: 1314-1320.[CrossRef][Medline]
Chiozzi P, Sanz JM, Ferrari D, Falzoni S, Aleotti A, Buell GN, Collo G, and Di Virgilio F (1997) Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor. J Cell Biol 138: 697-706.
Clyne JD, Wang LF, and Hume RI (2002) Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor. J Neurosci 22: 3873-3880.
Collo G, Neidhart S, Kawashima E, Kosco-Vilbois M, North RA, and Buell G (1997) Tissue distribution of the P2X7 receptor. Neuropharmacology 36: 1277-1283.[CrossRef][Medline]
Cucchiarini M, Ren XL, Perides G, and Terwilliger EF (2003) Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and type 5 vectors. Gene Ther 10: 657-667.[CrossRef][Medline]
Di Virgilio F, Chiozzi P, Falzoni S, Ferrari D, Sanz JM, Venketaraman V, and Baricordi OR (1998) Cytolytic P2X purinoceptors. Cell Death Differ 5: 191-199.[CrossRef][Medline]
Duan S, Anderson CM, Keung EC, Chen Y, and Swanson RA (2003) P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J Neurosci 23: 1320-1328.
Dubyak GR and el-Moatassim C (1993) Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol 265: C577-C606.
el-Moatassim C and Dubyak GR (1993) Dissociation of the pore-forming and phospholipase D activities stimulated via P2z purinergic receptors in BAC1.2F5 macrophages. Product inhibition of phospholipase D enzyme activity. J Biol Chem 268: 15571-15578.
Ennion SJ and Evans RJ (2002) Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface. Mol Pharmacol 61: 303-311.
Evans RJ, Lewis C, Buell G, Valera S, North RA, and Surprenant A (1995) Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol 48: 178-183.[Abstract]
Ferrari D, Stroh C, and Schulze-Osthoff K (1999) P2X7/P2Z purinoreceptor-mediated activation of transcription factor NFAT in microglial cells. J Biol Chem 274: 13205-10.
Ferrari D, Wesselborg S, Bauer MK, and Schulze-Osthoff K (1997) Extracellular ATP activates transcription factor NF-kappaB through the P2Z purinoreceptor by selectively targeting NF-kappaB p65. J Cell Biol 139: 1635-1643.
Humphreys BD, Virginio C, Surprenant A, Rice J, and Dubyak GR (1998) Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor homologues. Mol Pharmacol 54: 22-32.
Jiang LH, Mackenzie AB, North RA, and Surprenant A (2000a) Brilliant blue G selectively blocks ATP-gated rat P2X7 receptors. Mol Pharmacol 58: 82-88.
Jiang LH, Rassendren F, Surprenant A, and North RA (2000b) Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor. J Biol Chem 275: 34190-34196.
Khakh BS, Bao XR, Labarca C, and Lester HA (1999) Neuronal P2X transmittergated cation channels change their ion selectivity in seconds. Nat Neurosci 2: 322-330.[CrossRef][Medline]
Khakh BS, Burnstock G, Kennedy C, King BF, North RA, Séguéla P, Voigt M, and Humphrey PP (2001) International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol Rev 53: 107-118.
Lammas DA, Stober C, Harvey CJ, Kendrick N, Panchalingam S, and Kumararatne DS (1997) ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors. Immunity 7: 433-444.[CrossRef][Medline]
Le KT, Babinski K, and Séguéla P (1998) Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18: 7152-7159.
MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA, and Surprenant A (2001) Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 15: 825-835.[CrossRef][Medline]
Neary JT, Kang Y, Willoughby KA, and Ellis EF (2003) Activation of extracellular signal-regulated kinase by stretch-induced injury in astrocytes involves extracellular ATP and P2 purinergic receptors. J Neurosci 23: 2348-2356.
Nicke A, Baumert HG, Rettinger J, Eichele A, Lambrecht G, Mutschler E, and Schmalzing G (1998) P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO (Eur Mol Biol Organ) J 17: 3016-3028.[CrossRef][Medline]
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.
Smith RA, Alvarez AJ, and Estes DM (2001) The P2X7 purinergic receptor on bovine macrophages mediates mycobacterial death. Vet Immunol Immunopathol 78: 249-262.[CrossRef][Medline]
Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, and Gabel CA (2001) Altered cytokine production in mice lacking P2X7 receptors. J Biol Chem 276: 125-132.
Surprenant A, Rassendren F, Kawashima E, North RA, and Buell G (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science (Wash DC) 272: 735-738.[Abstract]
Torres GE, Egan TM, and Voigt MM (1999) Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. J Biol Chem 274: 6653-6659.
Virginio C, Church D, North RA, and Surprenant A (1997) Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36: 1285-1294.[CrossRef][Medline]
Virginio C, MacKenzie A, North RA, and Surprenant A (1999a) Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor. J Physiol 519: 335-346.
Virginio C, MacKenzie A, Rassendren FA, North RA, and Surprenant A (1999b) Pore dilation of neuronal P2X receptor channels. Nat Neurosci 2: 315-321.[CrossRef][Medline]
Wang ZZ, Mathias A, Gautam M, and Hall ZW (1999) Metabolic stabilization of muscle nicotinic acetylcholine receptor by rapsyn. J Neurosci 19: 1998-2007.
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) and C168A (
) mutants. HEK293 cells were transfected with a fixed concentration of wild-type P2X7 cDNA and varying concentrations of the mutant subunits. The solid and dashed lines represent the expected dose-response curves assuming trimeric and tetrameric channel configuration, respectively.
