Analysis of Assembly and Trafficking of Native P2X4 and P2X7 Receptor Complexes in Rodent Immune Cells

P2X4 and P2X7 are the predominant P2X receptor subtypes expressed in immune cells. Having previously shown a structural and functional interaction between the two recombinant receptors, our aims here were to identify the preferred assembly pathway of the endogenous receptors in macrophage-like cells and to investigate the trafficking of these receptors between the plasma membrane and intracellular sites. We exploited the difference in size between the two subunits, and we used a combination of cross-linkers and blue native-PAGE analysis to investigate the subunit composition of complexes present in primary cultures of rat microglia and macrophages from wild type and P2X7^–/– mice. Our results indicate that the preferred assembly pathway for both receptors is the formation of homotrimers. Homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. In both macrophages and microglia, P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular. There were clear cell type-dependent differences in the extent to which P2X4 receptors trafficked to and from the surface; trafficking was much more dynamic in microglia than in the macrophages, and further activation of cultured microglia with relatively short (3-h) incubations with lipopolysaccharide caused an ∼4-fold increase in the fraction of receptors at the surface with only a 1.2-fold increase in total expression. The redistribution of intracellular receptors is thus an efficient means of enhancing the functional expression of P2X4 at the plasma membrane of microglia.Acting via P2X receptors, ATP has several effects on immune cells, including triggering the release of pro-inflammatory cytokines, programmed cell death, and mycobacterial killing (1, 2). Until recently, attention has focused on the P2X7 receptor as the relevant sensor at the cell surface, and there is evidence for its involvement in neuropathic and inflammatory pain (3), arthritis (4), and the control of mycobacterial infection (5). P2X7 has a low affinity for ATP compared with the other predominant subtype expressed by immune cells, P2X4. The role of P2X4 receptors is less well understood, although in microglia they were shown to be up-regulated following peripheral nerve injury and to play an important role in the development of neuropathic pain (6). Regulation of the plasma membrane expression of these two receptors is not understood. Also, despite the considerable interest in both these receptor subtypes as potential targets in development of novel pain therapies, the subunit composition of the native receptors has not been determined.P2X receptor subunits associate to form trimeric complexes around a central conduction pore (7, 8). With the exception of P2X6, they form functional homomeric receptors, and several subtypes also form functional heterotrimers (9). There is also evidence that P2X receptors form larger signaling complexes, interacting with other neighboring P2X receptors and also with ion channels that belong to different structural classes, including members of the Cys loop receptor family (10) and the gap junction family (11). Higher order structures, with molecular mass corresponding to hexamers and nonamers, have been identified for heterologously expressed P2X2 and P2X4 receptors, suggesting that two or three receptors can form a stable association (12). Consistent with this idea, P2X receptor channels within a patch of membrane have been shown to open in a non-independent manner and to display positive cooperativity (13, 14).P2X4 and P2X7 are frequently co-expressed, not only in immune cells but also in epithelia and endothelia (15). The P2X7 subtype differs from other members of the family in that it has a very long cytoplasmic C-terminal tail, a low affinity toward ATP, is preferentially activated by BzATP, and couples to the opening of a pore permeable to large molecules up to 900 Da (2). P2X4 receptors have considerably higher affinity for ATP; they are up-regulated by the allosteric modulator ivermectin and are sensitive to the antagonist TNP-ATP³ at micromolar concentrations. It has been widely assumed that P2X7 does not form heteromeric assemblies with other members of the P2X family; however, recent evidence has indicated a structural and functional interaction between P2X4 and P2X7 receptors. First, P2X receptor currents recorded from airway-ciliated cells were reported to show a combination of P2X4-like and P2X7-like properties (16). Second, we showed that P2X4 and P2X7 could be co-immunoprecipitated both from mouse bone marrow-derived macrophages (BMDMs) and also when heterologously co-expressed (17). In addition, we used two nonfunctional point mutants of P2X4 to demonstrate a functional interaction between the two receptors; one mutant exerted a dominant negative effect on P2X7 receptor currents, whereas another conferred P2X4-like properties on the whole cell currents, namely sensitivity to ivermectin and TNP-ATP. Our conclusion was that P2X4 and P2X7 form a heteromeric association but that it remained to be established whether or not they assemble as heterotrimers around the same conduction pore.In this study, we set out first to address the question of the preferred assembly pathway of native P2X4 and P2X7 receptors in immune cells, and second to compare how these receptors traffic within immune cells. We took advantage of the size difference between P2X4 and P2X7 subunits to analyze the composition of the predominant dimeric and trimeric forms. We found that in rodent macrophages and microglia, P2X4 and P2X7 receptors preferentially assemble as homotrimers. Comparison of the trafficking of these receptors indicated cell type-dependent differences in the extent to which the predominantly intracellular P2X4 receptor traffics to and from the surface.     

Expression of P2X receptors on immune cells in the rat liver during postnatal development

Single and double-labeling immunofluorescence and RT-PCR expression of P2X receptor proteins and mRNAs were used in a study of the liver of postnatal rats. OX62 and ED1 were used as markers for dendritic and macrophage (Kupffer) cells respectively. The results showed that the P2X₆ receptor subunit was up-regulated by 15-fold on hepatic sinusoid cells during postnatal days P1 to P60. Subpopulations of Kupffer cells co-expressed P2X₄ and P2X₆ receptor subunits and dendritic cells co-expressed P2X₄ and P2X₇ receptor subunits. Lipopolysaccharide (endotoxin) injected into the peritoneal cavity led to increased expression of the P2X₆ receptor on Kupffer cells, suggesting that the P2X₆ receptor subunit may be up-regulated by endotoxin. This study presents the first evidence that P2X receptors are widely distributed in the rat liver immune system and that activation of Kupffer and dendritic cells in the rat liver might be regulated by extracellular ATP.     

Identification of a tubulin binding motif on the P2X2 receptor

To isolate proteins interacting with P2X receptors, GST fusion proteins containing the intracellular C terminal tail of P2X(2), P2X(5), or P2X(7) were used as bait to screen detergent extracts of rat brain synaptosomes. By SDS-PAGE combined with mass spectrometry, two interacting proteins were identified: betaIII tubulin and myelin basic protein. While myelin basic protein bound to all three P2X subunits, betaIII tubulin interacted exclusively with the P2X(2) subunit. The tubulin binding domain could be confined to a proline-rich segment (amino acids 371-412) of the P2X(2) subunit. Our results suggest a role for microtubules in the cellular localisation of the P2X(2) receptor.     

Differential expression of ATP-gated P2X receptors in DRG between chronic neuropathic pain and visceralgia rat models

There are divergences between neuropathic pain and visceralgia in terms of the duration, location, and character of hyperalgesia. It is generally recognized that nociceptive receptors, including P2X receptors, may play different roles in nociceptive mechanisms. The different roles of P2X1–7 receptors have not been fully understood both in neuropathic pain and visceral hyperalgesia. In order to explore the different expressions of P2X1–7 receptors in these two hyperalgesia models, the lumbosacral dorsal root ganglion (DRG) neurons from rat sciatic nerve chronic constriction injury (CCI) model and neonatal colorectal distention (NCRD) model were studied (both the primary nociceptive neuron afferents of those two models projected to the same segment of spinal cord). Both immunohistochemistry (IHC) technique and real-time fluorescence quantitative polymerase chain reaction (RT-PCR) technology were applied to analyze the protein expression levels and nucleic acid of P2X1–7 receptors. We found that except P2X2 and P2X3, the expression levels of P2X1 and P2X5 receptors increased in neuropathic pain while those expression levels of P2X4, P2X6, and P2X7 receptors increased in visceral pain. Our results also suggested that in addition to P2X2/3 heteromeric, other P2X subunits may also involved in generation heteromeric such as P2X1/5 and/or P2X2/5 in neuropathic pain and P2X4/6 and/or P2X4/7 in visceral pain.     

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.     

Production of monomeric antigen-enzyme conjugate to study requirements for follicular immune complex trapping.

Studies concerning the localization of immune complexes in lymphoid follicles and the involvement of these trapped immune complexes in the regulation of the immune response have thus far been performed with poorly defined complexes in terms of size and composition. For that reason, the minimum requirements for trapping in terms of number of antigen- and antibody molecules present in immune complexes could not be determined. We here describe the production and in vivo use of a monomeric HSA-HRP antigen-enzyme conjugate, readily demonstrable in cryostat sections and ELISA. This conjugate was obtained by combining the glutaraldehyde coupling-method with chromatography to fractionate monomeric and multimeric constituents. SDS-PAGE analysis showed that the conjugate consisted of a single molecular species of 109 kDa, whereas the often used periodate oxidation coupling method yielded a heterogeneous population of multimeric, oligomeric and monomeric molecules. We investigated the minimal size requirements for the composition of immune complexes to be trapped in murine spleen follicles using three different conjugates (monomeric HSA-HRP, multimeric HSA-HRP and multimeric HSA-HRP-Penicillin) and a panel of anti-HSA and anti-Penicillin monoclonal antibodies. We demonstrate that the smallest immune complexes, consisting of one antibody and two conjugate molecules, do not localize in splenic follicles. Immune complexes prepared with a single monoclonal antibody localize in follicles only if the epitope recognized occurs repeatedly on the antigen. The relevance of these results for physiological follicular trapping of protein antigens is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.     

Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners

P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X1 through P2X7) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homo-oligomeric complexes, the exception being P2X6. When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X7. Whereas P2X1, P2X2, P2X5, and P2X6 were able to assemble with most subunits, P2X3 and P2X4 presented a more restricted pattern of co-association. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP.     

Involvement of the purinergic P2X7 receptor in the formation of multinucleated giant cells

Multinucleated giant cells (MGC), a hallmark of chronic inflammatory reactions, remain an enigma of cell biology. There is evidence implicating the purinergic P2X7 receptor in the fusion process leading to MGC. To investigate this, we used HEK 293 cells stably transfected with either 1) the full-length rat P2X7 receptor (P2X7 cells), 2) a rat P2X7 receptor lacking the C-terminal domain (P2X7TC), or 3) a mock vector, and rat alveolar macrophages (MA) expressing the native receptor. P2X7 cells cultured in serum-free medium formed increased numbers of MGC and displayed a higher fusion index compared with mock transfectants. Stimulation of P2X7 pore-forming activity in P2X7 cells by polymyxin B (PMB) further increased significantly the formation of MGC. Conversely, blockers of P2X-receptors including oxidized ATP, brilliant blue G, and pyridoxal phosphate-6-azophenyl-2'-4'-disulfonic acid inhibited significantly MGC formation in both unstimulated and PMB-stimulated P2X7-transfected cells. In contrast, cells transfected with the truncated P2X7TC were devoid of pore-forming activity, did not respond to PMB stimulation, and failed to form enhanced numbers of MGC, thus behaving as mock transfectants. As found for P2X7-transfected cells, PMB also potentiated dose-dependently the formation of multinucleated MA by rat alveolar MA. Pretreatment with oxidized ATP abrogated the PMB stimulatory effects. Together, these data demonstrate unequivocally the participation of P2X7 receptor in the process of MGC formation. Our study also provides evidence suggesting that stimulation of the P2X7 receptor pathway in MA may mediate increased formation of MGC during chronic inflammatory reactions.     

Identification of a domain involved in ATP-gated ionotropic receptor subunit assembly.

P2X receptors are ATP-gated ion channels found in a variety of tissues and cell types. Seven different subunits (P2X(1)-P2X(7)) have been molecularly cloned and are known to form homomeric, and in some cases heteromeric, channel complexes. However, the molecular determinants leading to the assembly of subunits into P2X receptors are unknown. To address this question we utilized a co-immunoprecipitation assay in which epitope-tagged deletion mutants and chimeric constructs were examined for their ability to co-associate with full-length P2X subunits. Deletion mutants of the P2X(2) receptor subunit were expressed individually and together with P2X(2) or P2X(3) receptor subunits in HEK 293 cells. Deletion of the amino terminus up to the first transmembrane domain (amino acid 28) and beyond (to amino acid 51) did not prevent subunit assembly. Analysis of the carboxyl terminus demonstrated that mutants missing the portion of the protein downstream of the second transmembrane domain could also still co-assemble. However, a mutant terminating 25 amino acids before the second transmembrane domain could not assemble with other subunits or itself, implicating the missing region of the protein in assembly. This finding was supported and extended by data utilizing a chimera strategy that indicated TMD2 is a critical determinant of P2X subunit assembly.     

Pepstatin-sensitive proteolytic activity of sorghum seeds

Two novel proteinases were isolated from resting sorghum seeds and purified 100-fold. The activity of the purified enzymes was completely inhibited by pepstatin A and was unaffected by PMSF, leupeptin, EDTA and E-64 (L-trans-epoxysuccinyl leucylamino 4 guanidino butane), which indicates that they belong to the class of aspartic proteinases. SDS-PAGE and native-PAGE revealed a monomeric 29-kDa enzyme and a heterodimeric 61-kDa enzyme with two S-S linked subunits of 49 and 12 kDa. The proteases have maximum activity at 45 °C and pH 3.5, with haemoglobin as substrate. Activity at 60 °C is higher than at 30 °C.     

Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging

Seven P2X purinergic receptor subunits have been identified: P2X1–P2X7. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits might interact. Here, His₆-tagged P2X2, HA-tagged P2X4 and FLAG-tagged P2X6 subunits were co-expressed in tsA 201 cells. After sequential co-immunoprecipitation using anti-HA and anti-FLAG beads, all three subunits were present, demonstrating their interaction. Atomic force microscopy (AFM) imaging revealed receptors that were specifically decorated by both an anti-His₆ antibody and an anti-HA Fab fragment, indicating the presence of a P2X2/4/6 heterotrimer. To our knowledge, this is the first report of a P2X receptor containing three different subunits.     

Production of monomeric antigen-enzyme conjugate to study requirements for follicular immune complex trapping

Summary Studies concerning the localization of immune complexes in lymphoid follicles and the involvement of these trapped immune complexes in the regulation of the immune response have thus far been performed with poorly defined complexes in terms of size and composition. For that reason, the minimum requirements for trapping in terms of number of antigen- and antibody molecules present in immune complexes could not be determined. We here describe the production and in vivo use of a monomeric HSA-HRP antigen-enzyme conjugate, readily demonstrable in cryostat sections and ELISA. This conjugate was obtained by combining the glutaraldehyde coupling-method with chromatography to fractionate monomeric and multimeric constituents.SDS-PAGE analysis showed that the conjugate consisted of a single molecular species of 109 kDa, whereas the often used periodate oxidation coupling method yielded a heterogeneous population of multimeric, oligomeric and monomeric molecules.We investigated the minimal size requirements for the composition of immune complexes to be trapped in murine spleen follicles using three different conjugates (monomeric HSA-HRP, multimeric HSA-HRP and multimeric HSA-HRP-Penicillin) and a panel of anti-HSA and anti-Penicillin monoclonal antibodies. We demonstrate that the smallest immune complexes, consisting of one antibody and two conjugate molecules, do not localize in splenic follicles. Immune complexes prepared with a single monoclonal antibody localize in follicles only if the epitope recognized occurs repeatedly on the antigen.The relevance of these results for physiological follicular trapping of protein antigens is discussed. The described method for the production of monomeric enzyme-labelled protein applicable in histochemistry and ELISA should prove useful to prepare other conjugates of defined size for studies of trapping and other applications.Abbreviations FDC follicular dendritic cell - HRP horseradish peroxidase - HSA human serum albumin - HY anti-HSA hyperimmune serum - MAb monoclonal antibody - Pen penicillin     

Changes in expression of P2 receptors in rat and mouse pancreas during development and ageing

In view of the evidence for a role for extracellular ATP in both pancreatic endocrine and exocrine functions, we have investigated the expression of P2X and P2Y receptors in this tissue in neonate and aged rat and mouse. Using immunohistochemistry it was shown that P2X(1), P2X(4), P2X(7), P2Y(1) and P2Y(2) receptors were present in different regions of the rat and mouse pancreas; P2X(3) and P2X(6) receptors were not found, and P2X(5) immunolabelling was only found in some nerves. The pancreatic vasculature of both rat and mouse expressed P2X(1), P2X(2), P2Y(1) and P2Y(2) receptors in the smooth muscle. P2X(1) and P2X(4) receptors were absent in the islets of the neonate pancreas, but were progressively upregulated with age after birth. In contrast, the greatest expression of P2Y(1) in cells from the duct system was in neonate pancreas, while there was no P2Y(1) expression in aged rat pancreas. P2X(7) receptors had a consistent pattern of distribution in all of the groups examined, being located in the outer periphery of the islet. Using antibodies raised against insulin, somatostatin and glucagon, double-labelling immunofluorescence was used to identify P2X(7)-positive cells in different islet of Langerhans cell populations. Our results demonstrated a clear immunoreaction to P2X(7) receptors in islet alpha cells, while no P2X(7) was expressed in beta and delta cells. The significance of the differential expression of P2 receptors in the pancreas during development and ageing, and a possible role for the proliferation and death of the islet cell population are discussed.     

Somatic and axonal effects of ATP via P2X2 but not P2X7 receptors in rat thoracolumbar sympathetic neurones

Excitatory ATP responses in rat cultured thoracolumbar sympathetic neurones are mediated by somatic P2X(2) receptors. The present study investigated a possible role of axonal P2X(2) as well as P2X(7) receptors on the same preparation. Confocal laser scanning microscopy demonstrated P2X(2) and P2X(7) immunoreactivity along the axons as well as P2X(7) immunoreactivity surrounding the cell nuclei. P2X(7) mRNA expression was detected in individual neurones using a single-cell RT-PCR approach. Adenosine triphosphate (ATP) caused a significant increase in axonal Ca(2+) concentration which was dependent on external Ca(2+) but insensitive to depletion of the cellular Ca(2+) pools by cyclopiazonic acid. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 micro m) virtually abolished the ATP response, whereas brilliant blue G (0.1 micro m), a selective P2X(7) receptor antagonist, had no effect. Dibenzoyl-ATP (BzATP; 100 micro m) induced a much smaller increase in axonal [Ca(2+)] concentration than ATP at equimolar concentrations. The response to BzATP was distinctly reduced by PPADS but not by brilliant blue G. The overall pharmacological profile of the axonal P2X receptors resembled closely that of the somatic P2X(2) receptors. In conclusion, the present data suggest the occurrence of axonal excitatory P2X(2) receptors in thoracolumbar sympathetic neurones. However, the functional significance of axonal and (peri)-nuclear P2X(7) receptors has still to be proven.     

Modulation of the humoral immune response by antibody-mediated antigen targeting to complement receptors and Fc receptors

During an ongoing immune response, immune complexes, composed of Ag, complement factors, and Igs, are formed that can interact with complement receptors (CRs) and IgG Fc receptors (Fc gamma R). The role of CR1/2 and Fc gamma R in the regulation of the immune response was investigated using OVA that was chemically conjugated to whole IgG of the rat anti-mouse CR1/2 mAb 7G6. FACS analysis using the murine B cell lymphoma IIA1.6 confirmed that the 7G6-OVA conjugate recognized CR1/2. Incubating IIA1.6 cells with 7G6-OVA triggered tyrosine phosphorylation and Ag presentation to OVA-specific T cells in vitro. Immunizing mice with 7G6-OVA at a minimal dose of 1 microgram i.p. per mouse markedly enhanced the anti-OVA Ig response, which was primarily of the IgG1 isotype subclass. The 7G6-OVA did not enhance the anti-OVA response in CR1/2-deficient mice. OVA coupled to an isotype control Ab induced a considerably lower anti-OVA response compared with that induced by OVA alone, suggesting inhibition by interaction between the Fc part of the Ab and the inhibitory Fc gamma RIIb on B cells. This findings was supported by the observation that IIA1.6 cells which were incubated with 7G6-OVA lost the ability to present Ag upon transfection with Fc gamma RIIb. In sum, 7G6-conjugated OVA, resembling a natural immune complex, induces an enhanced anti-OVA immune response that involves at least CR1/2-mediated stimulation and that may be partially suppressed by Fc gamma RIIb.     

Biochemical and functional evidence for heteromeric assembly of P2X1 and P2X4 subunits

P2X receptors are ligand-gated ion channels activated by extracellular ATP. In expression systems, P2X subunits form homo- and heterotrimeric receptors. Heteromerization is also likely to occur in vivo as (i) most P2X subunits show overlapping distribution in different tissues and (ii) the functional properties of many native P2X receptors differ from those of heterologously expressed homomeric receptors. Here, we used the Xenopus laevis oocyte expression system to test for heteromerization of P2X1 and P2X4 subunits. Upon co-injection, P2X4 subunits were co-purified with hexahistidyl-tagged P2X1 subunits indicating heteromerization. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis of these P2X complexes excluded artificial aggregation and confirmed that both subunits were present in trimeric complexes of the same size. Two-electrode voltage-clamp experiments revealed functional P2X receptors with kinetic properties resembling homomeric P2X4 receptors and a pharmacological profile similar to homomeric P2X1 receptors. Thus, application of alpha,beta-methylene ATP evoked a slowly desensitizing current sensitive to the antagonists suramin and 2',3'-O-(2,4,6-trinitrophenyl)-ATP. This study provides for the first time biochemical and functional evidence for the formation of heteromeric P2X(1+4) receptors. These receptors may account for native P2X mediated responses that until now could not be correlated with previously described recombinant P2X receptors.     

Distribution of P2X receptors in the rat adrenal gland

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.     

Regulation of phospholipase D by P2X7 receptors in submandibular ductal cells

ATP (1 mM) increased the phospholipase D (PLD) activity of rat submandibular gland (RSMG) ductal cells in a concentration-dependent and calcium-sensitive manner. The response to ATP was reproduced by benzoylbenzoyl-ATP (Bz-ATP, 100 microM) and also partly by adenosine 5'-(gamma-thio)triphosphate (ATPgammaS, 1 mM). A similar stimulation was observed in control mice (P2X7R+/+ mice) but not in mice lacking the P2X7 receptors (P2X7R-/- mice). Oxidized ATP and Coomassie blue or the addition of magnesium or nickel to the incubation medium inhibited the response to ATP. The stimulation of PLD by purinergic agonist was inhibited by about 50% by calphostin C and chelerythrine, two protein kinase C (PKC) inhibitors. The stimulation of PLD by Bz-ATP and by o-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester which activates PKC, were not additive.From these results we can conclude that the activation of P2X7 receptors in RSMG ductal cells is coupled to the activation of a PLD. This activation is partly mediated by protein kinase C.     

The P2X7 carboxyl tail is a regulatory module of P2X7 receptor channel activity

P2X(7) receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X(7) subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X(7) subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434-595) in Xenopus laevis oocytes. P2X(7) channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only approximately 5% of the full-length P2X(7) receptor current. Co-assembly of channel core subunits with full-length P2X(7) subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X(7) receptor activity. Co-expression of the tail extension as a discrete protein increased ATP-gated current amplitudes of P2X(7) channel cores 10-20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X(7) receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X(7) channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X(7) subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409-436 and 434-494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X(7) channel gating sensitive to intracellular regulation.