Human P2X7 Receptor Research Articles

Cryo-EM structures of the human P2X1 receptor reveal subtype-specific architecture and antagonism by supramolecular ligand-binding

P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design.

Structural insights into the human P2X1 receptor and ligand interactions.

The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions, offering the potential for new drug treatments. However, progress in this area has been hindered by limited structural information and a lack of well-characterised tool compounds. In this study, we employ cryogenic electron microscopy (cryo-EM) to elucidate the structures of the P2X1 receptor in an ATP-bound desensitised state and an NF449-bound closed state. NF449, a potent P2X1 receptor antagonist, engages the receptor distinctively, while ATP, the endogenous ligand, binds in a manner consistent with other P2X receptors. To explore the molecular basis of receptor inhibition, activation, and ligand interactions, key residues involved in ligand and metal ion binding were mutated. Radioligand binding assays with [3H]-α,β-methylene ATP and intracellular calcium ion influx assays were used to evaluate the effects of these mutations. These experiments validate key ligand-receptor interactions and identify conserved and non-conserved residues critical for ligand binding or receptor modulation. This research expands our understanding of the P2X1 receptor structure at a molecular level and opens new avenues for in silico drug design targeting the P2X1 receptor.

Discovery of a Potent, Orally Active, and Long-Lasting P2X7 Receptor Antagonist as a Preclinical Candidate for Delaying the Progression of Chronic Kidney Disease.

Chronic kidney disease (CKD) is a condition characterized by functional deterioration with sustained inflammation and progressive fibrosis of the kidneys affecting over 800 million people worldwide. The P2X7 receptor (P2X7R) plays a key role in CKD progression. Our previous P2X7R antagonists demonstrated good efficacy for treating kidney injury but were limited by low oral exposure and short half-life, restricting their application. This study reports the optimization of P2X7R antagonists for better oral pharmacokinetics. The candidate compound 13a with the respective IC50 of 34.86 and 25.28 nM against human and murine P2X7R, administered orally at 10 mg/kg in mice, exhibits a remarkably long half-life of 161.64 h, with a high exposure of 1,163,980.55 μg·h/L. Oral administration of 13a (0.3 or 1.0 mg/kg, twice weekly) significantly reduced renal injury and fibrosis in unilateral ureteral obstruction and adenine diet-induced mice models, highlighting its potential for delaying the progression of CKD.

Exploring P2X receptor activity: A journey from cellular impact to electrophysiological profiling

The development of in vitro pharmacological assays relies on creating genetically modified cell lines that overexpress the target protein of interest. However, the choice of the host cell line can significantly impact the experimental outcomes. This study explores the functional characterization of P2X7 and P2X4 receptor modulators through cellular assays and advanced electrophysiological techniques. The influence of different host cell lines (HEK-293, HEK-293FT, and 1321N1) on the activity of reference agonists and antagonists targeting human and murine P2X4 and P2X7 receptors was systematically investigated, highlighting the significant impact of the host cell on experimental results. The 1321N1 cell line was identified as the preferred host cell line when investigating the human P2X4 receptor due to more consistent agonist activities, antagonist potencies, and a more stable assay signal window. Furthermore, a patch-clamp protocol that allows for the repetitive recording of ATP-mediated inward currents from isolated human CD4+ T-cells was established, revealing that both P2X7 and P2X4 receptors are crucial for immune cell regulation, positioning them as promising therapeutic targets for managing inflammatory disorders.

Two serial filters control P2X7 cation selectivity, Ser342 in the central pore and lateral acidic residues at the cytoplasmic interface.

The human P2X7 receptor (hP2X7R) is a homotrimeric cell surface receptor gated by extracellular ATP4- with two transmembrane helices per subunit, TM1 and TM2. A ring of three S342 residues, one from each pore-forming TM2 helix, located halfway across the membrane bilayer, functions to close and open the gate in the apo and ATP4--bound open states, respectively. The hP2X7R is selective for small inorganic cations, but can also conduct larger organic cations such as Tris+. Here, we show by voltage-clamp electrophysiology in Xenopus laevis oocytes that mutation of S342 residues to positively charged lysines decreases the selectivity for Na+ over Tris+, but maintains cation selectivity. Deep in the membrane, laterally below the S342 ring are nine acidic residues arranged as an isosceles triangle consisting of residues E14, D352, and D356 on each side, which do not move significantly during gating. When the E14K mutation is combined with lysine substitutions of D352 and/or D356, cation selectivity is lost and permeation of the small anion Cl- is allowed. Lysine substitutions of S342 together with D352 or E14 plus D356 in the acidic triangle convert the hP2X7R mutant to a fully Cl--selective ATP4--gated receptor. We conclude that the ion selectivity of wild-type hP2X7R is determined by two sequential filters in one single pathway: (i) a primary size filter, S342, in the membrane center and (ii) three cation filters lateral to the channel axis, one per subunit interface, consisting of a total of nine acidic residues at the cytoplasmic interface.

Human P2X4 receptor gating is modulated by a stable cytoplasmic cap and a unique allosteric pocket.

P2X receptors (P2XRs) are a family of ATP-gated ion channels comprising homomeric and heteromeric trimers of seven subunits (P2X 1 - P2X 7 ) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes the stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-EM structures of full-length, wild-type human P2X 4 receptor in apo, antagonist-bound, and desensitized states. Because the apo and antagonist-bound structures of this slow-desensitizing P2XR include an intact cytoplasmic cap while the desensitized state structure does not, the cytoplasmic cap forms before agonist binding. Furthermore, structural and functional data suggests the cytoplasmic cap is stabilized by lipids to slow desensitization and that P2X 4 is further modified by glycosylation and palmitoylation. Finally, our antagonist-bound inhibited state structure reveals features specific to the allosteric ligand-binding pocket in human receptors that empower the development of small-molecule modulators.

Preparation and preliminary evaluation of a tritium-labeled allosteric P2X4 receptor antagonist.

P2X4 receptors are ATP-gated cation channels that were proposed as novel drug targets due to their role in inflammation and neuropathic pain. Only few potent and selective P2X4 receptor antagonists have been described to date. Labeled tool compounds suitable for P2X4 receptor binding studies are lacking. Here, we present a novel allosteric P2X4 receptor antagonist possessing high potency in the low nanomolar range. We describe its tritium-labeling resulting in the P2X4-selective radiotracer [3H]PSB-OR-2020 with high specific activity (45 Ci/mmol; 1.67 TBq/mmol). A radioligand binding assay was developed using human embryonic kidney (HEK293) cell membranes recombinantly expressing the human P2X4 receptor. Competition binding studies with structurally diverse P2X4 receptor antagonists revealed different allosteric binding sites indicating that the new class of P2X4 receptor antagonists, to which PSB-OR-2020 belongs, interacts with an unprecedented allosteric site. [3H]PSB-OR-2020 may become a useful tool for research on P2X4 receptors and for promoting drug development.

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.

Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

BackgroundThe purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC.Materials and methodsBlood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay.ResultsRisk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1β, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects.ConclusionIn conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.

Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells.

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies.

The human P2X7 receptor alters microglial morphology and cytokine secretion following immunomodulation.

Introduction: In recent years, purinergic signaling via the P2X7 receptor (P2X7R) on microglia has repeatedly been implicated in depression genesis. However, it remains unclear which role the human P2X7R (hP2X7R) plays in regulating both microglia morphology and cytokine secretion upon different environmental and immune stimuli, respectively. Methods: For this purpose, we used primary microglial cultures derived from a humanized microglia-specific conditional P2X7R knockout mouse line to emulate different gene-environment interactions between microglial hP2X7R and molecular proxies of psychosocial and pathogen-derived immune stimuli. Microglial cultures were subjected to treatments with the agonists 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP) and lipopolysaccharides (LPS) combined with specific P2X7R antagonists (JNJ-47965567, A-804598). Results: Morphotyping revealed overall high baseline activation due to the in vitro conditions. Both BzATP and LPS + BzATP treatment increased round/ameboid microglia and decreased polarized and ramified morphotypes. This effect was stronger in hP2X7R-proficient (CTRL) compared to knockout (KO) microglia. Aptly, we found antagonism with JNJ-4796556 and A-804598 to reduce round/ameboid microglia and increase complex morphologies only in CTRL but not KO microglia. Single cell shape descriptor analysis confirmed the morphotyping results. Compared to KO microglia, hP2X7R-targeted stimulation in CTRLs led to a more pronounced increase in microglial roundness and circularity along with an overall higher decrease in aspect ratio and shape complexity. JNJ-4796556 and A-804598, on the other hand, led to opposite dynamics. In KO microglia, similar trends were observed, yet the magnitude of responses was much smaller. Parallel assessment of 10 cytokines demonstrated the proinflammatory properties of hP2X7R. Following LPS + BzATP stimulation, IL-1β, IL-6, and TNFα levels were found to be higher and IL-4 levels lower in CTRL than in KO cultures. Vice versa, hP2X7R antagonists reduced proinflammatory cytokine levels and increased IL-4 secretion. Discussion: Taken together, our results help disentangle the complex function of microglial hP2X7R downstream of various immune stimuli. In addition, this is the first study in a humanized, microglia-specific in vitro model identifying a so far unknown potential link between microglial hP2X7R function and IL-27 levels.

Diclofenac and other non-steroidal anti-inflammatory drugs (NSAIDs) are competitive antagonists of the human P2X3 receptor.

Introduction: The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products, and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents. Methods: To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology. Results: We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 and 76.7µM, respectively). A weaker inhibition of hP2X1R, hP2X4R, and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) inhibited hP2X3R, rP2X3R, and hP2X7R (IC50 values of 221µM, 264.1µM, and ∼900µM, respectively), calling into question its use as a non-selective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP application or increasing concentrations of the agonist α,β-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results suggest a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains, inhibits the gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R, and hP2X7R. Discussion: Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac, which are rarely reached in the therapeutic range, may play a minor role in analgesia compared to the high-potency cyclooxygenase inhibition but may explain the known side effect of taste disturbances caused by diclofenac.

Activation of endothelial NO synthase and P2X7 receptor modification mediates the cholinergic control of ATP-induced interleukin-1β release by mononuclear phagocytes.

The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a central role in host defense against infections. High systemic IL-1β levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1β release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1β release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1β was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. The inhibitory effect of nAChR agonists on the BzATP-induced IL-1β release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1β release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1β release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1β release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

Conformational dynamics of the cytoplasmic cap during desensitization of the hP2X3R.

The human P2X3 receptor (P2X3R) is a trimeric ligand-gated ion channel activated by extracellular ATP. X-ray crystallography revealed atomic structures of its apo (closed, resting), ATP-bound (open, activated), and ATP-bound (closed, desensitized) states. An intracellular ‘cytoplasmic cap’ was found to stabilize the open pore in the ATP-bound state. However, the cytoplasmic cap was not resolved in the resting or desensitized structures, suggesting a model in which transient formation of the cytoplasmic cap is associated with the opening of the ion pore. To study the functional coupling between (un)folding of the cytoplasmic cap and opening/closing of the P2X3R ion pore, we performed extensive all-atom molecular dynamics (MD) simulations combined with voltage-clamp electrophysiology and mutagenesis. Temperature replica-exchange MD simulations of P2X3R revealed the cytoplasmic cap's unfolding pathway and consequent ion pore closure. Our results show that the N- and C-terminal layers that form the cytoplasmic cap in the open state do not fully unfold during desensitization. By contrast, the partial unfolding of the lower C-terminal layer is sufficient to initiate pore closure/desensitization. In contrast, the N-terminal layers remain folded and keep their spatial proximity to the membrane. Interestingly, not all C-terminal secondary structures unfold, and some structural organization persists, which may allow for rapid transitions between the apo, open, and resting states. The main trigger of pore closure/desensitization is the loss of contact between the C-terminal and the N-terminal part of the cap. We investigated point mutations, aimed at (de)stabilizing the cytoplasmic cap, using free-energy calculations and electrophysiological recordings. These demonstrate that the kinetics of P2X3R desensitization depends on the folding-free energy of the cytoplasmic cap. In summary, our work reveals an allosteric coupling mechanism between the cytoplasmic cap and gating of the transmembrane ion pore in P2X receptors.

Identification of residues involved in homotrimeric assembly of the human P2X4 receptor by simulations and site-directed mutagenesis.

P2X receptors (P2XRs) are a family of ATP-gated cation channels with seven members, P2X1-P2X7. P2XRs are involved in numerous physiological and pathophysiological functions and are therefore interesting drug targets. Functional P2XRs are obligate homotrimers or heterotrimers of three identical or homologous subunits, respectively, with each subunit having a large ectodomain flanked by two membrane-spanning helices, TM1 and TM2, and N- and C-terminal intracellular domains.

Extracellular binding sites of positive and negative allosteric P2X4 receptor modulators

AimsP2X receptors are ATP-gated ion channels which play a role in many pathophysiological conditions. They are considered as novel drug targets, particularly in the fields of pain, (neuro) inflammation, and cancer. Due to difficulties in developing drug-like orthosteric ligands that bind to the highly polar ATP binding site, the design of positive and negative allosteric modulators (PAMs and NAMs) is a promising strategy. The P2X4 receptor was proposed as a novel target for neuropathic and inflammatory pain (antagonists), and for the treatment of alcoholism (PAMs). So far, little is known about the allosteric binding site(s) of P2X4 receptors. The aim of this study was to identify the binding site(s) of the macrocyclic natural product ivermectin, the urea derivative BX430, and the antidepressant drug paroxetine that act as allosteric modulators of P2X4 receptors. Material and methodsWe generated chimeric receptors in which extracellular sequences of the human P2X4 receptor were exchanged for corresponding residues of the human P2X2 receptor, complemented by specific single amino acid residue mutants. Chimeric and mutated receptors were stably expressed in 1321N1 astrocytoma cells, and characterized by fluorimetric measurement of ATP-induced Ca2+-influx. In addition, docking studies utilizing a homology model of the human P2X4 receptor were performed. Key findingsOur results suggest a common binding site for ivermectin and BX430 in an extracellular receptor domain, while paroxetine might bind to the cation pore. SignificanceThe obtained results provide a basis for the development of positive and negative allosteric P2X4 modulators with improved properties and will support future drug development efforts.

Characterization of a microglia-specific humanized P2X7 receptor knock-out mouse line: Implications for translational psychoneuroimmunology

IntroductionDepression is a common psychiatric disorder and chronic stress is considered its main environmental risk factor. Recently, immune processes including adenosine triphosphate mediated P2X7 receptor (P2X7R) signalling via microglia and macrophages (M/Ms) were found to play a critical role in depression genesis, by linking environmental stress to depression biology and symptoms.ObjectivesTo characterize the role of human P2X7R (hP2X7R) in psychosocial and immune stress conditions, both in vitro and in vivo.MethodsSeveral, custom designed mouse lines expressing the loxP-flanked, hP2X7R-sequence in the murine P2X7R locus were established. In addition, these mice possess a Cre-sensitive reporter and express a Cre recombinase fused to a mutant estrogen receptor ligand-binding domain in M/Ms. This enables conditional, tamoxifen-inducible hP2X7R inactivation and simultaneous tdTomato expression. First, we established primary microglia cell cultures and characterized them at baseline and following immune stimulation. Next, we performed behavioural assessment of hP2X7Rwt and microglia-specific hP2X7RKO mice following chronic psychosocial stress. Last, we developed a novel in vivo two-photon microscopy (TPM) approach by use of frontolimbic cranial windows.ResultsPrimary hP2X7RKO microglia displayed significantly lower IL-1β production, increased survival and decreased morphological activation upon immune stimulation. Although hP2X7RKO mice showed a significant increase of locomotor activity at baseline, there was no impact on anxiety- and depressive-like phenotypes. Longitudinal in vivo TPM enabled morphometric characterization of cortical M/Ms over several weeks.ConclusionsOur results illustrate the great potential of this humanized mouse line for translational psychiatry. In the future, this system could proof useful to evaluate immunomodulatory approaches in chronic stress and depression.DisclosureNo significant relationships.

Medullary astrocytes mediate irregular breathing patterns generation in chronic heart failure through purinergic P2X7 receptor signalling.

SummaryBackgroundBreathing disorders (BD) (apnoeas/hypopneas, periodic breathing) are highly prevalent in chronic heart failure (CHF) and are associated with altered central respiratory control. Ample evidence identifies the retrotrapezoid nucleus (RTN) as an important chemosensitivity region for ventilatory control and generation of BD in CHF, however little is known about the cellular mechanisms underlying the RTN/BD relationship. Within the RTN, astrocyte‐mediated purinergic signalling modulates respiration, but the potential contribution of RTN astrocytes to BD in CHF has not been explored.MethodsSelective neuron and/or astrocyte-targeted interventions using either optogenetic and chemogenetic manipulations in the RTN of CHF rats were used to unveil the contribution of the RTN on the development/maintenance of BD, the role played by astrocytes in BD and the molecular mechanism underpinning these alterations.FindingsWe showed that episodic photo-stimulation of RTN neurons triggered BD in healthy rats, and that RTN neurons ablation in CHF animals eliminates BD. Also, we found a reduction in astrocytes activity and ATP bioavailability within the RTN of CHF rats, and that chemogenetic restoration of normal RTN astrocyte activity and ATP levels improved breathing regularity in CHF. Importantly, P"X/ P2X7 receptor (P2X7r) expression was reduced in RTN astrocytes from CHF rats and viral vector-mediated delivery of human P2X7 P2X7r into astrocytes increases ATP bioavailability and abolished BD.InterpretationOur results support that RTN astrocytes play a pivotal role on BD generation and maintenance in the setting CHF by a mechanism encompassing P2X7r signalling.FundingThis study was funded by the National Research and Development Agency of Chile (ANID).

Elevated Serum Purine Levels in Schizophrenia: A Reverse Translational Study to Identify Novel Inflammatory Biomarkers.

BackgroundImmunological markers and related signaling molecules in the blood are altered in schizophrenia mouse models, in acutely relapsed patients with schizophrenia, and in persons at a clinically high risk for subsequently developing psychosis, highlighting their potential as prognostic and theranostic biomarkers. Therefore, we herein aimed to identify novel potential biomarkers in the serum that are associated with purinergic signaling.MethodsTo our knowledge, this is the first study to assess the correlations among the levels of human serum adenine nucleotides (ATP, ADP), adenosine, P2X7 receptor, and disease activity in patients hospitalized due to an acute relapse of schizophrenia (n = 53) and healthy controls (n = 47). In addition, to validate these findings using a reverse translational approach, we examined the same parameters in an acute phencyclidine-induced schizophrenia mouse model.ResultsWe found consistently elevated levels of ATP, ADP, interleukin (IL)-6, and IL-10 in both schizophrenia groups compared with the controls. The levels of adenosine, IL-1β, IL-12, and C-reactive protein were also increased in the human patient samples. Moreover, ATP and ADP were significantly positively correlated with the Positive and Negative Symptom Scale item “lack of judgment and insight”; IL-1β, IL-12, and tumour necrosis factor alpha were significantly positively correlated with “tension” and “depression”; and “disorientation” and “poor attention” were correlated significantly with IL-6 and IL-8.ConclusionsOur study suggests the promising potential of blood purines and inflammatory markers as future prognostic tools.