Mouse P2X7 Research Articles

Pharmacological differences between human and mouse P2X4 receptor explored using old and new tools

There is growing interest in the P2X4 receptor as a therapeutic target for several cardiovascular, inflammatory and neurological conditions. Key to exploring the physiological and pathophysiological roles of P2X4 is access to selective compounds to probe function in cells, tissues and animal models. There has been a recent growth in selective antagonists for P2X4, though agonist selectivity is less well studied. As there are some known pharmacological differences between P2X receptors from different species, it is important to understand these differences when designing a pharmacological strategy to probe P2X4 function in human tissue and mouse models. Here, we provide a systematic comparison of agonist and antagonist pharmacology in 1321N1 cells expressing either human or mouse P2X4 orthologues. We identify a rank order of agonist potency of ATP > 2-MeSATP > αβmeATP = BzATP > CTP = γ-[(propargyl)-imido]-ATP for human P2X4 and ATP > 2-MeSATP = CTP > ATPγS = γ-[(propargyl)-imido]-ATP = BzATP for mouse. Human P2X4 is not activated by ATPγS but can be activated by αβmeATP. We identify a rank order of antagonist potency of BAY-1797 = PSB-12062 = BX-430 > 5-BDBD > TNP-ATP = PPADS for human P2X4 and BAY-1797 > PSB-12062 = PPADS > TNP-ATP for mouse. Mouse P2X4 is not antagonised by 5-BDBD or BX-430. The study reveals key pharmacological differences between human and mouse P2X4, highlighting caution when selecting tools for comparative studies between human and mouse and ascribing cellular responses of some commonly used agonists to P2X4.

Site-Directed Mutagenesis Reveals P2XR7 Extracellular Domain Residues That Facilitate Channel Activation by Histone Proteins

Seven mammalian ionotropic purinergic receptors (P2XRs) have been identified (P2XR1-7). All P2XRs have a large extracellular domain containing a conserved extracellular ATP binding pocked, however, P2XRs vary widely in ATP affnity and desensitization kinetics. P2XR7 is unique in that it has the lowest ATP affnity (mM), does not desensitize to ATP, and has a large intracellular C-terminal domain. In our recent work, we demonstrated that histone proteins activate P2XR7 current. However, histone-evoked currents are not inhibited by selective inhibitors of P2XR7 ATP-evoked current. We hypothesized that histone proteins may bind to a distinct allosteric site in the P2XR7 extracellular domain. To test this, we expressed mouse and human P2X receptors (m.- and h.P2XR1-7) in heterologous cells (Xenopus oocytes) and measured inward cation currents using a two-electrode voltage clamp (TEVC). This allowed us to determine which P2XR channels are suffcient for histone-induced currents and provided a strategy to determine the P2XR7 residues involved in histone- and ATP-evoked currents. Histone proteins induced large inward cation currents in oocytes expressing m.P2XR7 and m.P2XR4, but not in those expressing m.P2XR1. Because histone proteins are highly alkaline, we aligned the extracellular domains of m.P2XR7, -4, and -1, and looked for acidic residues that were conserved in P2XR7 and -4 but absent in P2XR1. This approach identified 12 possible histone binding residues, of which three (D156N, E175K, and E240Q) were found to be silent mutations. Mutations at these sites decreased histone-evoked current but did not decrease ATP-evoked current. These results provide functional insight into a new allosteric mechanism of P2XR7 channel activation by histone proteins which may be used to guide the development of new pharmacologic and therapeutic strategies. NIH R00HL133451 and R01HL155180. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Virtual screening indicates potential inhibitors of the P2X7 receptor

Anti-inflammatory agents can be synthetic or natural compounds and are often used to attenuate different levels of inflammation. Inflammatory diseases, due to the involvement of multiple systems, are becoming difficult to treat, involve long durations of therapy where applicable, have a high cost of management and have a deleterious impact on public health. The search for natural and synthetic compounds with anti-inflammatory activity is an important strategy in drug design. Bioactive synthetic drugs may be repurposed for other pharmacological applications, and natural product chemical structures offer unlimited opportunities for new drug discoveries due to the unparalleled availability of chemical diversity. Virtual screening of 2774 molecules on the mouse P2X7 protein showed that potential ligands are composed of five flavonoids (narirutin, diosmin, complanatuside, hesperidin, and oroxin B) and other drugs such as velpatasvir, itacitinib and lifitegrast. In vitro studies in mouse cells confirmed the inhibitory activity of the indicated ligands on the P2X7 receptor by applying virtual screening. The behavior of protein bonded to the ligands was verified by analysis of the molecular dynamic simulation trajectories for four of the most potent inhibitor compounds, indicating that the ligands velpatasvir, itacitinib, lithospermic acid and narirutin remained in the binding site indicated by molecular docking.

Taspine is a natural product that suppresses P2X4 receptor activity via phosphoinositide 3‐kinase inhibition

P2X4 is a ligand-gated cation channel activated by extracellular ATP involved in neuropathic pain, inflammation and arterial tone. Natural products were screened against human or mouse P2X4 activity using fura-2 loaded 1321N1 cells for measurement of intracellular Ca2+ responses. Whole-cell currents were measured by patch clamp. Human primary macrophage chemokine release was used to assess effect of taspine on inflammatory cell function. An enzymatic assay was performed to assess the effect of taspine on recombinant PI3-kinase. A natural product screen identified taspine as an inhibitor of human P2X4 activity. Taspine inhibits human and mouse P2X4-mediated Ca2+ influx in 1321N1 cells expressing receptors but lacked activity at human P2X2, P2X3, P2X2/3 and P2X7 receptors. Taspine inhibited the maximal response at human and mouse P2X4 but effective on ATP potency. Taspine has a slow onset rate (~15 min for half-maximal inhibition), irreversible over 30 min of washout. Taspine inhibits P2X4-mediated Ca2+ signalling in mouse BV-2 microglia cells and human primary macrophage. Taspine inhibited P2X4-mediated CXCL5 secretion in human primary macrophage. Taspine reversed ivermectin-induced potentiation of P2X4 currents in 1321N1 stably expressing cells. The PI3-kinase inhibitor LY294002 mimicked the properties of taspine on P2X4-mediated Ca2+ influx and whole-cell currents. Taspine directly inhibited the enzymatic activity of recombinant PI3-kinase in a competitive manner. Taspine is a novel natural product P2X4 receptor inhibitor, mediating its effect through PI3-kinase inhibition rather than receptor antagonism. Taspine can inhibit the pro-inflammatory signalling by P2X4 in human primary macrophage.

Structural and Functional Features of the P2X4 Receptor: An Immunological Perspective.

Extracellular nucleotides are important mediators of activation, triggering various responses through plasma membrane P2 and P1 receptors. P2 receptors are further subdivided into ionotropic P2X receptors and G protein-coupled P2Y receptors. P2X4 is an ATP-gated cation channel broadly expressed in most tissues of the body. Within the P2X family, P2X4 has a unique subcellular distribution, being preferentially localized in lysosomes. In these organelles, high ATP concentrations do not trigger P2X4 because of the low pH. However, when the pH increases to 7.4, P2X4 can be stimulated by intra-lysosomal ATP, which is in its active, tetra-anionic form. Elucidation of P2X4, P2X3 and P2X7 structures has shed some light on the functional differences between these purinergic receptors. The potential interaction between P2X4 and P2X7 has been extensively studied. Despite intensive effort, it has not been possible yet to determine whether P2X4 and P2X7 interact as heterotrimers or homotrimers at the plasma membrane. However, several publications have shown that functional interactions between P2X4 and P2X7 do occur. Importantly, these studies indicate that P2X4 potentiates P2X7-dependent activation of inflammasomes, leading to increased release of IL-1β and IL-18. The role of P2X4 in various diseases could be beneficial or deleterious even though the pathophysiological mechanisms involved are still poorly defined. However, in diseases whose physiopathology involves activation of the NLRP3 inflammasome, P2X4 was found to exacerbate severity of disease. The recent production of monoclonal antibodies specific for the human and mouse P2X4, some of which are endowed with agonist or antagonist properties, raises the possibility that they could be used therapeutically. Analysis of single nucleotide polymorphisms of the human P2RX4 gene has uncovered the association of P2RX4 gene variants with susceptibility to several human diseases.

Antibodies binding the head domain of P2X4 inhibit channel function and reverse neuropathic pain.

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.

Design, synthesis and evaluation in an LPS rodent model of neuroinflammation of a novel 18F-labelled PET tracer targeting P2X7

BackgroundThe P2X7 receptor has been shown to play a fundamental role in the initiation and sustenance of the inflammatory cascade. The development of a novel fluorine-18 PET tracer superior and with a longer half-life to those currently available is a promising step towards harnessing the therapeutic and diagnostic potential offered by this target. Inspired by the known antagonist A-804598, the present study outlines the design via molecular docking, synthesis and biological evaluation of the novel P2X7 tracer [18F]EFB. The tracer was radiolabelled via a three-step procedure, in vitro binding assessed in P2X7-transfected HEK293 and in B16 cells by calcium influx assays and an initial preclinical evaluation was performed in a lipopolysaccharide (LPS)-injected rat model of neuroinflammation.ResultsThe novel tracer [18F]EFB was synthesised in 210 min in 3–5% decay-corrected radiochemical yield (DC RCY), >99% radiochemical purity (RCP) and >300 GBq/μmol and fully characterised. Functional assays showed that the compound binds with nM Ki to human, rat and mouse P2X7 receptors. In vivo, [18F]EFB displayed a desirable distribution profile, and while it showed low blood–brain barrier penetration, brain uptake was quantifiable and displayed significantly higher mean longitudinal uptake in inflamed versus control rat CNS regions.Conclusions[18F]EFB demonstrates strong in vitro affinity to human and rodent P2X7 and limited yet quantifiable BBB penetration. Considering the initial promising in vivo data in an LPS rat model with elevated P2X7 expression, this work constitutes an important step in the development of a radiotracer useful for the diagnosis and monitoring of clinical disorders with associated neuroinflammatory processes.

Species Difference in Sensitivity of Human and Mouse P2X7 Receptors to Inhibitory Effects of Divalent Metal Cations.

P2X7 receptor (P2X7R), a purinergic receptor, is involved in pathophysiological events such as inflammation and cell death, and thus is an attractive target for therapeutic approaches. It is reported that divalent metal cations (DMCs) inhibit P2X7R activation and that there are species differences in their inhibitory effects. To extrapolate the findings in experimental animals to humans, these species differences have to be clarified, but species differences in the sensitivity of P2X7R to DMCs between man and mouse have not been demonstrated. Here we performed direct comparison of the inhibitory effects of DMCs on human and mouse P2X7R activation. Cell lines constitutively expressing human and mouse P2X7R were used, and their P2X7R activation was evaluated as means of YO-PRO-1 dye uptake. MgCl2, NiCl2, ZnCl2, CuCl2 and CaCl2 dose-dependently decreased agonist-induced YO-PRO-1 uptake via both human and mouse P2X7Rs. Apparent differences in the inhibitory profiles for NiCl2 and CaCl2 between them were found, and the IC50 values of DMCs were in the order of CaCl2>MgCl2>NiCl2≈ZnCl2>CuCl2 for both human and mouse P2X7Rs. In this study, we demonstrate that human P2X7R exhibits different sensitivity to nickel and calcium compared with the case of the mouse one, while there is no species difference in the sensitivity of their P2X7Rs to magnesium, zinc and copper, suggesting that the effects of magnesium, zinc and copper on P2X7R-associated pathophysiological events in humans might be predicted from those in mice.

Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation.

Ion channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5'-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1β (IL-1β). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1β release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeutics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7.

Characterization of P2X4 receptor agonists and antagonists by calcium influx and radioligand binding studies

Antagonists for ATP-activated P2X4 ion channel receptors are currently in the focus as novel drug targets, in particular for the treatment of neuropathic and inflammatory pain. We stably expressed the human, rat and mouse P2X4 receptors in 1321N1 astrocytoma cells, which is devoid of functional nucleotide receptors, by retroviral transfection, and established monoclonal cell lines. Calcium flux assay conditions were optimized for high-throughput screening resulting in a Z′-factor of >0.8. The application of ready-to-use frozen cells did not negatively affect the results of the calcium assays, which is of great advantage for the screening of compound libraries. Species differences were observed, the rat P2X4 receptor being particularly insensitive to many ATP derivatives. Membrane preparations of the cell lines showed high levels of specific [35S]ATPγS binding with low nonspecific binding (<5% of total binding), while non-transfected cells were devoid of specific binding sites for the radioligand. Conditions were employed which allow binding studies to be performed at room temperature. While a variety of nucleotide-derived agonists and the antagonist TNP-ATP displaced [35S]ATPγS from its binding site at human P2X4 receptors, the non-nucleotidic antagonists paroxetine and 5-BDBD did not compete with radioligand binding and were therefore characterized as allosteric antagonists. Homology modeling was applied to find an explanation for the observed species differences.

Nucleoside Reverse Transcriptase Inhibitors Suppress Laser-Induced Choroidal Neovascularization in Mice.

To evaluate the efficacy of nucleoside reverse transcriptase inhibitors (NRTIs) in the laser-induced mouse model of choroidal neovascularization (CNV). We evaluated the NRTIs lamivudine (3TC), zidovudine (AZT), and abacavir (ABC) and the P2X7 antagonist A438079. Choroidal neovascularization was induced by laser injury in C57BL/6J wild-type, Nlrp3-/-, and P2rx7-/- mice, and CNV volume was measured after 7 days by confocal microscopy. Drugs were administered by intravitreous injection immediately after the laser injury. Vascular endothelial growth factor-A in RPE-choroid lysates was measured 3 days after laser injury by ELISA. HEK293 cells expressing human and mouse P2X7 were exposed to the selective P2X7 receptor agonist 2', 3'-(benzoyl-4-benzoyl)-ATP (Bz-ATP) with or without 3TC, and VEGF-A levels in media were measured by ELISA. Intravitreous injection of 3TC, AZT, and ABC significantly suppressed laser-induced CNV in C57BL/6J wild-type and Nlrp3-/- mice (P < 0.05) but not in P2rx7-/- mice. Intravitreous injection of A438079 also suppressed the laser-induced CNV (P < 0.05). The NRTIs 3TC, AZT, and ABC blocked VEGF-A levels in the RPE/choroid after laser injury in wild-type (P < 0.05) but not P2rx7-/- mice. Moreover, there was no additive effect of 3TC on CNV inhibition when coadministered with a neutralizing VEGF-A antibody. Stimulation of human and mouse P2X7-expressing HEK293 cells with Bz-ATP increased VEGF secretion (P < 0.001), which was abrogated by 3TC (P < 0.001). Stimulation of primary human RPE cells with Bz-ATP increased VEGFA and IL6 mRNA levels, which were abrogated by 3TC. Multiple clinically relevant NRTIs suppressed laser-induced CNV and downregulated VEGF-A, via P2X7.

P2X7 receptor antagonist activity of the anti-allergic agent oxatomide

Activation of the P2X7 receptor by extracellular ATP is associated with various immune responses including allergic inflammation. Anti-allergic agents, such as H1-antihistamines, are known to inhibit the effects of different chemical mediators such as acetylcholine and platelet-activating factor. Therefore, we hypothesized that some anti-allergic agents might affect P2X7 receptor function. Using N18TG2 and J774 cells, which express functional P2X7 receptors, the effects of several anti-allergic agents on P2X7 receptor function were investigated by monitoring the ATP-induced increase in intracellular Ca2+ concentrations ([Ca2+]i). Among the various agents tested, oxatomide significantly inhibited P2X7 receptor-mediated [Ca2+]i elevation in a concentration-dependent manner without affecting the P2Y2 receptor-mediated response in both N18TG2 and J774 cells. Consistently, oxatomide inhibited P2X7 receptor-mediated membrane current and downstream responses such as mitogen-activated protein kinase activation, inflammation-related gene induction, and cell death. In addition, oxatomide inhibited P2X7 receptor-mediated degranulation in mouse bone marrow-derived mast cells. Whole cell patch clamp analyses in HEK293 cells expressing human, mouse, and rat P2X7 receptors revealed that the inhibitory effect of oxatomide on ATP-induced current was most prominent for the human P2X7 receptor and almost non-existent for the rat P2X7 receptor. The potent inhibitory effects of oxatomide on human P2X7 receptor-mediated function were confirmed in RPMI8226 human B cell-like myeloma cells, which endogenously express the P2X7 receptor. Our results demonstrated that the antihistamine oxatomide also acts as a P2X7 receptor antagonist. Future studies should thus evaluate whether P2X7 receptor antagonism contributes to the anti-allergic effects of oxatomide.

Identification and Characterization of a Selective Allosteric Antagonist of Human P2X4 Receptor Channels

P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.

Tanshinone II A sulfonate, but not tanshinone II A, acts as potent negative allosteric modulator of the human purinergic receptor P2X7.

Tanshinone II A sulfonate (TIIAS) was identified as a potent, selective blocker of purinergic receptor P2X7 in a compound library screen. In this study, a detailed characterization of the pharmacologic effects of TIIAS on P2X7 is provided. Because TIIAS is a derivative of tanshinone II A (TIIA) and both compounds have been used interchangeably, TIIA was included in some assays. Fluorometric and electrophysiologic assays were used to characterize effects of TIIAS and TIIA on recombinantly expressed human, rat, and mouse P2X7. Results were confirmed in human monocyte-derived macrophages expressing native P2X7. In all experiments, involvement of P2X7 was verified using established P2X7 antagonists. TIIAS, but not TIIA, reduces Ca(2+) influx via human P2X7 (hP2X7) with an IC50 of 4.3 µM. TIIAS was less potent at mouse P2X7 and poorly inhibited rat P2X7. Monitoring of YO-PRO-1 uptake confirmed these findings, indicating that formation of the hP2X7 pore is also suppressed by TIIAS. Electrophysiologic experiments revealed a noncompetitive mode of action. TIIAS time-dependently inhibits hP2X7 gating, possibly by binding to the intracellular domain of the receptor. Inhibition of native P2X7 in macrophages by TIIAS was confirmed by monitoring Ca(2+) influx, YO-PRO-1 uptake, and release of the proinflammatory cytokine interleukin-1β. Fluorometric experiments involving recombinantly expressed rat P2X2 and human P2X4 were conducted and verified the compound's selectivity. Our data suggest that hP2X7 is a molecular target of TIIAS, but not of TIIA, a compound with different pharmacologic properties.

Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin.

P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current-voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.

The P2X7 receptor channel: recent developments and the use of P2X7 antagonists in models of disease.

The P2X7 receptor is a trimeric ATP-gated cation channel found predominantly, but not exclusively, on immune cells. P2X7 activation results in a number of downstream events, including the release of proinflammatory mediators and cell death and proliferation. As such, P2X7 plays important roles in various inflammatory, immune, neurologic and musculoskeletal disorders. This review focuses on the use of P2X7 antagonists in rodent models of neurologic disease and injury, inflammation, and musculoskeletal and other disorders. The cloning and characterization of human, rat, mouse, guinea pig, dog, and Rhesus macaque P2X7, as well as recent observations regarding the gating and permeability of P2X7, are discussed. Furthermore, this review discusses polymorphic and splice variants of P2X7, as well as the generation and use of P2X7 knockout mice. Recent evidence for emerging signaling pathways downstream of P2X7 activation and the growing list of negative and positive modulators of P2X7 activation and expression are also described. In addition, the use of P2X7 antagonists in numerous rodent models of disease is extensively summarized. Finally, the use of P2X7 antagonists in clinical trials in humans and future directions exploring P2X7 as a therapeutic target are described.

The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor

P2X7, an ATP-gated cation channel, is involved in immune cell activation, hyperalgesia and neuropathic pain. By regulating cytokine release in the brain, P2X7 has been linked to the pathophysiology of mood disorders and schizophrenia. We here assess the impact of 123 drugs that act in the central nervous system on human P2X7. Most prominently, the tricyclic antipsychotics prochlorperazine (PCP) and trifluoperazine (TFP) potently inhibited P2X7-mediated Ca2+ entry, dye permeation and ionic currents. In divalent cation-containing bath solutions or after prolonged incubation, ATP-evoked P2X7 currents were inhibited by 10 μM PCP. This effect was not related to dopamine receptor antagonism. Surprisingly, PCP co-applied with ATP enhanced inward currents in bath solutions with low divalent cation concentrations. Intracellular perfusion with PCP did not substitute for the extracellularly applied drug, indicating that its binding sites are accessible from the extracellular space. Since P2X7 current potentiation by PCP was voltage-dependent, at least one site may be located within the electrical field of the membrane. While the channel opening and closure kinetic was altered by PCP, the apparent affinity of ATP remained unchanged (potentiation) or changed slightly (inhibition). Measurements in human monocyte-derived macrophages confirmed the PCP-induced inhibition of ATP-evoked Ca2+ influx, Yo-Pro-1 permeability, and whole cell currents. Interestingly, neither heterologously expressed rat or mouse P2X7 nor native P2X7 in rat astrocyte cultures or in mouse bone marrow-derived macrophages were inhibited by perazines with a similar potency. We conclude that perazine-type neuroleptics are potent, but species-selective allosteric modulators of human but not murine P2X7 receptors.

Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors

In mammalian cells, the anti-parasitic drug ivermectin is known as a positive allosteric modulator of the ATP-activated ion channel P2X4 and is used to discriminate between P2X4- and P2X7-mediated cellular responses. In this paper we provide evidence that the reported isoform selectivity of ivermectin is a species-specific phenomenon. Complementary electrophysiological and fluorometric methods were applied to evaluate the effect of ivermectin on recombinantly expressed and on native P2X7 receptors. A biophysical characterization of ionic currents and of the pore dilation properties is provided. Unexpectedly, ivermectin potentiated currents in human monocyte-derived macrophages that endogenously express hP2X7 receptors. Likewise, currents and [Ca(2+) ](i) influx through recombinant human (hP2X7) receptors were potently enhanced by ivermectin at submaximal or saturating ATP concentrations. Since intracellular ivermectin did not mimic or prevent its activity when applied to the bath solution, the binding site of ivermectin on hP2X7 receptors appears to be accessible from the extracellular side. In contrast to currents through P2X4 receptors, ivermectin did not cause a delay in hP2X7 current decay upon ATP removal. Interestingly, NMDG(+) permeability and Yo-Pro-1 uptake were not affected by ivermectin. On rat or mouse P2X7 receptors, ivermectin was only poorly effective, suggesting a species-specific mode of action. The data indicate a previously unrecognized species-specific modulation of human P2X7 receptors by ivermectin that should be considered when using this cell-biological tool in human cells and tissues.

Expression, assembly and function of novel C‐terminal truncated variants of the mouse P2X7 receptor: re‐evaluation of P2X7 knockouts

Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7(-/-) mice (Pfizer KO). Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function.

Splice-variants of the P2X7 receptor reveal differential agonist-dependence and functional coupling with pannexin-1

P2X7 receptors function as ATP-gated cation channels but also interact with other proteins as part of a larger signalling complex to mediate a variety of downstream responses that are dependent upon the cell type in which they are expressed. Receptor-mediated membrane permeabilization to large molecules precedes the induction of cell death, but remains poorly understood. The mechanisms that underlie differential sensitivity to NAD are also unknown. By studying alternative variants of the mouse P2X7 receptor we show that sensitivity to NAD is mediated through the P2X7k variant, which has a much more restricted distribution than the P2X7a receptor, but is expressed in T lymphocytes. The altered N-terminus and TM1 of the P2X7k receptor enhances the stability of the active state of this variant compared with P2X7a, thereby increasing the efficacy of NAD-dependent ADP ribosylation as measured by ethidium uptake, a rise in intracellular Ca(2+) and the activation of inward currents. Co-expression of P2X7k and P2X7a receptors reduced NAD sensitivity. P2X7k-receptor-mediated ethidium uptake was also triggered by much lower BzATP concentrations and was insensitive to the P451L single nucleotide polymorphism. P2X7k-receptor-mediated ethidium uptake occurred independently of pannexin-1 suggesting a pathway intrinsic to the receptor. Only for the P2X7aL451 receptor could we resolve a component of dye uptake dependent upon pannexin-1. Signalling occurred downstream of the activation of caspases rather than involving direct cross talk between the channels. However, an in situ proximity assay showed close association between P2X7 receptors and pannexin-1, which would facilitate ATP efflux through pannexin-1 acting in an autocrine manner.