ATP-gated P2X Receptors Research Articles

On P2X receptors in the brain: microvessels. Dedicated to the memory of the late Professor Geoffrey Burnstock (1929-2020).

This tribute article presents selected immunocytochemical and transmission electron microscope data on the location of ATP-gated P2X receptor in the rat brain, as studied in the 1990s in Prof G. Burnstock's laboratory at University College London. There are examples of immuno-ultrastructural findings and introductory information about pre- and post-synaptic location of P2X receptors in the rat cerebellum and endocrine hypothalamus to support the concept of purinergic transmission in the central nervous system. Then findings of diverse immunoreactivity for P2X1, P2X2, P2X4, and P2X6 receptors associated with brain microvessels are shown, including vascular endothelium and pericytes as well as perivascular astrocytes and neuronal components. These findings imply the involvement of P2X receptors and hence purinergic signalling in the neurovascular unit, at least in microvessels in the rat cerebellum and hypothalamic paraventricular and supraoptic nuclei examined here. Various aspects of P2X receptors in brain microvessels are discussed.

Antagonism of the ATP-gated P2X7 receptor: a potential therapeutic strategy for cancer.

The P2X receptor 7 (P2X7R) is a plasma membrane receptor sensing extracellular ATP associated with a wide variety of cellular functions. It is most commonly expressed on immune cells and is highly upregulated in a number of human cancers where it can play a trophic role in tumorigenesis. Activation of this receptor leads to the formation of a non-selective cation channel, which has been associated with several cellular functions mediated by the PI3K/Akt pathway and protein kinases. Due to its broad range of functions, the receptor represents a potential therapeutic target for a number of cancers. This review describes the range of mechanisms associated with P2X7R activation in cancer settings and highlights the potential of targeted inhibition of P2X7R as a therapy. It also describes in detail a number of key P2X7R antagonists currently in pre-clinical and clinical development, including oxidised ATP, Brilliant Blue G (BBG), KN-62, KN-04, A740003, A438079, GSK1482160, CE-224535, JNJ-54175446, JNJ-55308942, and AZ10606120. Lastly, it summarises the in vivo studies and clinical trials associated with the use and development of these P2X7R antagonists in different disease contexts.

Ectonucleotidases in Acute and Chronic Inflammation.

Ectonucleotidases are extracellular enzymes with a pivotal role in inflammation that hydrolyse extracellular purine and pyrimidine nucleotides, e.g., ATP, UTP, ADP, UDP, AMP and NAD+. Ectonucleotidases, expressed by virtually all cell types, immune cells included, either as plasma membrane-associated or secreted enzymes, are classified into four main families: 1) nucleoside triphosphate diphosphohydrolases (NTPDases), 2) nicotinamide adenine dinucleotide glycohydrolase (NAD glycohydrolase/ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1), 3) ecto-5′-nucleotidase (NT5E), and 4) ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs). Concentration of ATP, UTP and NAD+ can be increased in the extracellular space thanks to un-regulated, e.g., cell damage or cell death, or regulated processes. Regulated processes include secretory exocytosis, connexin or pannexin hemichannels, ATP binding cassette (ABC) transporters, calcium homeostasis modulator (CALMH) channels, the ATP-gated P2X7 receptor, maxi-anion channels (MACs) and volume regulated ion channels (VRACs). Hydrolysis of extracellular purine nucleotides generates adenosine, an important immunosuppressant. Extracellular nucleotides and nucleosides initiate or dampen inflammation via P2 and P1 receptors, respectively. All these agents, depending on their level of expression or activation and on the agonist concentration, are potent modulators of inflammation and key promoters of host defences, immune cells activation, pathogen clearance, tissue repair and regeneration. Thus, their knowledge is of great importance for a full understanding of the pathophysiology of acute and chronic inflammatory diseases. A selection of these pathologies will be briefly discussed here.

Sphingosine-1-phosphate induces migration of microglial cells via activation of volume-sensitive anion channels, ATP secretion and activation of purinergic receptors

Microglia cells are versatile players coordinating inflammatory and regenerative processes in the central nervous system in which sphingosine-1-phosphate (S1P)-mediated migration is essential. We investigated the involved signaling cascade by means of voltage clamp, measurement of ATP secretion, and wound healing assay in murine microglial BV-2 cells. S1P and extracellular hypoosmolar solution evoked an anion conductance of the cell membrane. The corresponding ion currents were inhibited by intracellular hypoosmolar solution and by the anion channel antagonists NPPB, tamoxifen, and carbenoxolone, pointing to the activation of volume-regulated anion channels (VRAC). The knockdown by siRNA indicates the involvement of LRRC8A subunits. The S1PR1-antagonist W123 and pertussis-toxin prevented the S1P-induced currents, showing the involvement of the Gi-protein-coupled S1P receptor 1 (S1PR1). Furthermore, S1P and hypoosmolar extracellular solution induced an increase of ATP levels in the supernatants of BV-2 cells, which was inhibited by NPPB, tamoxifen, and W123. S1P, ATP, and ADP stimulated cell migration into the scratch area. The inhibition of S1PR1 and the downstream Gi proteins hampered cell migration. Antagonists of VRAC were also able to diminish the migration of BV-2 cells. Furthermore, direct inhibition of ATP-gated P2X4 or P2X7 receptors or ADP-stimulated P2Y12 receptors blocked the stimulating effects of S1P on BV-2 cell migration. We conclude that there is an interaction between S1P receptors and purinergic receptors mediated by an S1P-induced ATP release via VRAC and that the amount of released ATP is capable of stimulating cell migration of BV-2 microglia cells via activation of P2X4, P2X7, and P2Y12 receptors.

Deviant reporter expression and P2X4 passenger gene overexpression in the soluble EGFP BAC transgenic P2X7 reporter mouse model

The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases. P2X7 effects were also described in neurons and astrocytes but its localisation and function in these cell types has been challenging to demonstrate in situ. BAC transgenic mouse lines have greatly advanced neuroscience research and two BAC-transgenic P2X7 reporter mouse models exist in which either a soluble EGFP (sEGFP) or an EGFP-tagged P2X7 receptor (P2X7-EGFP) is expressed under the control of a BAC-derived P2rx7 promoter. Here we evaluate both mouse models and find striking differences in both P2X expression levels and EGFP reporter expression patterns. Most remarkably, the sEGFP model overexpresses a P2X4 passenger gene and sEGFP shows clear neuronal localisation but appears to be absent in microglia. Preliminary functional analysis in a status epilepticus model suggests functional consequences of the observed P2X receptor overexpression. In summary, an aberrant EGFP reporter pattern and possible effects of P2X4 and/or P2X7 protein overexpression need to be considered when working with this model. We further discuss reasons for the observed differences and possible caveats in BAC transgenic approaches.

Structural and Functional Basis for Understanding the Biological Significance of P2X7 Receptor.

The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels—inexistent in physiological conditions—are required for the receptor to be triggered and contribute to its role in cell damage signaling. The inconsistent data on its activation pathways and the few studies performed in natively expressed human P2X7R have led us to review the structure, activation pathways, and specific cellular location of P2X7R in order to analyze its biological relevance. The ATP-gated P2X7R is a homo-trimeric receptor channel that is occasionally hetero-trimeric and highly polymorphic, with at least nine human splice variants. It is localized predominantly in the cellular membrane and has a characteristic plasticity due to an extended C-termini, which confers it the capacity of interacting with membrane structural compounds and/or intracellular signaling messengers to mediate flexible transduction pathways. Diverse drugs and a few endogenous molecules have been highlighted as extracellular allosteric modulators of P2X7R. Therefore, studies in human cells that constitutively express P2X7R need to investigate the precise endogenous mediator located nearby the activation/modulation domains of the receptor. Such research could help us understand the possible physiological ATP-mediated P2X7R homeostasis signaling.

Chronic administration of P2X7 receptor antagonist JNJ-47965567 delays disease onset and progression, and improves motor performance in ALS SOD1G93A female mice.

ABSTRACTNeuroinflammation is one of the main physiopathological mechanisms of amyotrophic lateral sclerosis (ALS), produced by the chronic activation of microglia in the CNS. This process is triggered by the persistent activation of the ATP-gated P2X7 receptor (P2RX7, hereafter referred to as P2X7R). The present study aimed to evaluate the effects of the chronic treatment with the P2X7R antagonist JNJ-47965567 in the development and progression of ALS in the SOD1G93A murine model. SOD1G93A mice were intraperitoneally (i.p.) injected with either 30 mg/kg of JNJ-47965567 or vehicle 4 times per week, from pre-onset age (here, postnatal day 60; P60) until study endpoint. Body weight, motor coordination, phenotypic score, disease onset and survival were measured throughout the study, and compared between vehicle- and drug-injected groups. Treatment with the P2X7R antagonist JNJ-47965567 delayed disease onset, reduced body weight loss and improved motor coordination and phenotypic score in female SOD1G93A mice, although it did not increase lifespan. Interestingly, neither beneficial nor detrimental effects were observed in males in any of the analyzed parameters. Treatment did not affect motor neuron survival or ChAT, Iba-1 and P2X7R protein expression in endpoint individuals of mixed sexes. Overall, chronic administration of JNJ-47965567 for 4 times per week to SOD1G93A mice from pre-onset stage altered disease progression in female individuals while it did not have any effect in males. Our results suggest a partial, yet important, effect of P2X7R in the development and progression of ALS.

P2X7 Receptor Activity Limits Accumulation of T Cells within Tumors

Extracellular ATP (eATP) is a signaling molecule that variably affects all cells of the immune system either directly or after hydrolysis to adenosine. Although eATP is virtually absent in the interstitium of normal tissues, it can be present in the hundreds of micromolar range in tumors, a concentration compatible with activation of the ATP-gated ionotropic P2X7 receptor. Here, we show that P2X7 activity in tumor-infiltrating lymphocytes (TIL) induces cellular senescence and limits tumor suppression. P2X7 stimulation affected cell cycling of effector T cells and resulted in generation of mitochondrial reactive oxygen species and p38 MAPK-dependent upregulation of cyclin-dependent kinase inhibitor 1A (Cdkn1a, encoding for p21Waf1/Cip1). Lack of P2X7 promoted a transcriptional signature that correlated with enhanced cytotoxic T-cell response in human solid tumors. In mice, transfer of tumor-specific T cells with deletion of P2rx7 significantly reduced tumor growth and extended survival. Collectively, these findings uncover a purinergic checkpoint that can be targeted to improve the efficacy of cancer immunotherapy strategies. SIGNIFICANCE: These findings suggest that the purinergic checkpoint P2X7 may be targeted to enhance T-cell-mediated cancer immunotherapy and improve T effector cell accumulation in the tumor microenvironment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3906/F1.large.jpg.

Characterization of the Expression of the ATP-Gated P2X7 Receptor Following Status Epilepticus and during Epilepsy Using a P2X7-EGFP Reporter Mouse.

Mounting evidence suggests that the ATP-gated P2X7 receptor contributes to increased hyperexcitability in the brain. While increased expression of P2X7 in the hippocampus and cortex following status epilepticus and during epilepsy has been repeatedly demonstrated, the cell type-specific expression of P2X7 and its expression in extra-hippocampal brain structures remains incompletely explored. In this study, P2X7 expression was visualized by using a transgenic mouse model overexpressing P2X7 fused to the fluorescent protein EGFP. The results showed increased P2X7-EGFP expression after status epilepticus induced by intra-amygdala kainic acid and during epilepsy in different brain regions including the hippocampus, cortex, striatum, thalamus and cerebellum, and this was most evident in microglia and oligodendrocytes. Co-localization of P2X7-EGFP with cell type-specific markers was not detected in neurons or astrocytes. These data suggest that P2X7 activation is a common pathological hallmark across different brain structures, possibly contributing to brain inflammation and neurodegeneration following acute seizures and during epilepsy.

Protective Effects of Hydrogen Sulfide Against the ATP-Induced Meningeal Nociception.

We previously showed that extracellular ATP and hydrogen sulfide (H2S), a recently discovered gasotransmitter, are both triggering the nociceptive firing in trigeminal nociceptors implicated in migraine pain. ATP contributes to meningeal nociception by activating the P2X3 subunit-containing receptors whereas H2S operates mainly via TRP receptors. However, H2S was also proposed as a neuroprotective and anti-nociceptive agent. This study aimed to test the effect of H2S on ATP-mediated nociceptive responses in rat meningeal afferents and trigeminal neurons and on ATP-induced degranulation of dural mast cells. Electrophysiological recording of trigeminal nerve activity in meninges was supplemented by patch-clamp and calcium imaging studies of isolated trigeminal neurons. The H2S donor NaHS induced a mild activation of afferents and fully suppressed the subsequent ATP-induced firing of meningeal trigeminal nerve fibers. This anti-nociceptive effect of H2S was specific as an even stronger effect of capsaicin did not abolish the action of ATP. In isolated trigeminal neurons, NaHS decreased the inward currents and calcium transients evoked by activation of ATP-gated P2X3 receptors. Moreover, NaHS prevented ATP-induced P2X7 receptor-mediated degranulation of meningeal mast cells which emerged as triggers of migraine pain. Finally, NaHS decreased the concentration of extracellular ATP in the meningeal preparation. Thus, H2S exerted the multiple protective actions against the nociceptive effects of ATP. These data highlight the novel pathways to reduce purinergic mechanisms of migraine with pharmacological donors or by stimulation production of endogenous H2S.

P2X7 Receptor-Dependent microRNA Expression Profile in the Brain Following Status Epilepticus in Mice

The ionotropic ATP-gated P2X7 receptor is an important contributor to inflammatory signaling cascades via the release of Interleukin-1β, as well as having roles in cell death, neuronal plasticity and the release of neurotransmitters. Accordingly, there is interest in targeting the P2X7 receptor for the treatment of epilepsy. However, the signaling pathways downstream of P2X7 receptor activation remain incompletely understood. Notably, recent studies showed that P2X7 receptor expression is controlled, in part, by microRNAs (miRNAs). Here, we explored P2X7 receptor-dependent microRNA expression by comparing microRNA expression profiles of wild-type (wt) and P2X7 receptor knockout mice before and after status epilepticus. Genome-wide microRNA profiling was performed using hippocampi from wt and P2X7 receptor knockout mice following status epilepticus induced by intra-amygdala kainic acid. This revealed that the genetic deletion of the P2X7 receptor results in distinct patterns of microRNA expression. Specifically, we found that in vehicle-injected control mice, the lack of the P2X7 receptor resulted in the up-regulation of 50 microRNAs and down-regulation of 35 microRNAs. Post-status epilepticus, P2X7 receptor deficiency led to the up-regulation of 44 microRNAs while 13 microRNAs were down-regulated. Moreover, there was only limited overlap among identified P2X7 receptor-dependent microRNAs between control conditions and post-status epilepticus, suggesting that the P2X7 receptor regulates the expression of different microRNAs during normal physiology and pathology. Bioinformatic analysis revealed that genes targeted by P2X7 receptor-dependent microRNAs were particularly overrepresented in pathways involved in intracellular signaling, inflammation, and cell death; processes that have been repeatedly associated with P2X7 receptor activation. Moreover, whereas genes involved in signaling pathways and inflammation were common among up- and down-regulated P2X7 receptor-dependent microRNAs during physiological and pathological conditions, genes associated with cell death seemed to be restricted to up-regulated microRNAs during both physiological conditions and post-status epilepticus. Taken together, our results demonstrate that the P2X7 receptor impacts on the expression profile of microRNAs in the brain, thereby possibly contributing to both the maintenance of normal cellular homeostasis and pathological processes.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

TMEM163 Regulates ATP-Gated P2X Receptor and Behavior

Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and invivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators invivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs invivo and a potential target for the discovery of drugs for treating pain.

Astaxanthin Attenuates Neuroinflammation in Status Epilepticus Rats by Regulating the ATP-P2X7R Signal.

BackgroundAs a life-threatening neurological emergency, status epilepticus (SE) is often refractory to available treatment. Current studies have shown a causal role of neuroinflammation in patients with lower seizure thresholds and driving seizures. The ATP-gated purinergic P2X7 receptor (P2X7R) is mainly expressed on the microglia, which function as gatekeepers of inflammation. Although emerging evidence has demonstrated significant anti-inflammatory effects of astaxanthin (AST) in SE, the associated mechanism remains unclear. Therefore, this study aimed to clarify the effects of AST on P2X7R-related inflammation in SE.MethodsSE was induced in rats using lithium–pilocarpine, and AST was administered 1 h after SE induction. Rat microglia were treated with lipopolysaccharide (LPS), AST, ATP, 2,3-O-4-benzoyl-4-benzoyl-ATP (BzATP) and oxidized ATP (oxATP). The Morris water maze, immunohistochemistry, and Nissl staining were performed in rats. Expressions of P2X7R and inflammatory cytokines (such as cycloxygenase-2 (Cox-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α)) were detected using real-time polymerase chain reaction (RT-PCR) and Western blot (WB) both in rats and microglia. ATP concentration in the microglia was evaluated using ELISA.ResultsThe AST alleviated hippocampal injury and improved cognitive dysfunction induced by SE. AST also effectively inhibited inflammation and downregulated P2X7R expression in both rat brain and microglia. The results also showed that AST reduced the extracellular ATP levels and that P2X7R expression could be increased by extracellular ATP. In addition, BzATP upregulates the expression of P2X7R and inflammatory factors in microglia. Conversely, it downregulates the expression of P2X7R and inflammatory factors.ConclusionOur study suggests that AST attenuated ATP-P2X7R mediated inflammation in SE.

Association of Hypomorphic P2X7 Receptor Genotype With Age.

One of the main risk factors for brain diseases is aging. Recent studies have shown that aging is a progressive degenerative process associated with chronic low-level inflammation. The ATP-gated P2X7 receptor (P2X7R) plays an important role in inflammation and has been associated with different brain (e.g., Alzheimer’s and Parkinson’s) or other age-related (osteoporosis, arthritis, cancer) diseases. Several single nucleotide polymorphisms (SNPs) in the P2RX7 gene have been identified, including the loss-of-function 1513A>C and 1405A>G SNPs, and the gain-of-function 489C>T and 1068G>A SNPs. We carried out a literature analysis to verify an association between P2RX7 SNPs’ frequency and age. In 34 worldwide eligible studies (11.858 subjects) no correlation between 1513CC genotype frequency and age emerged. On the contrary, analysis of European Caucasian cohorts (7.241 subjects) showed a significant increase in 1513CC frequency with age (P = 0.027). In agreement with these findings, analysis of two publicly available datasets, including USA Caucasian cohorts, unveiled an increased frequency of 1513CC and 489CC genotypes with age (P = 0.0055 and P = 0.0019, respectively). Thus, hypomorphic P2RX7 genotypes may be positively selected with age in European and North American Caucasian populations. We hypothesize that Caucasian individuals bearing an anti-inflammatory P2X7R phenotype and living in high-income countries may have a longer life expectancy.

Differential Expression of the Metabotropic P2Y Receptor Family in the Cortex Following Status Epilepticus and Neuroprotection via P2Y1 Antagonism in Mice.

Purinergic signaling via P2 receptors is now widely accepted to play a critical role during increased states of hyperexcitability and seizure-induced pathology. In the setting of seizures and epilepsy, most attention has been paid to investigating the fast-acting ATP-gated P2X receptor family. More recent evidence has now also provided compelling evidence of an involvement of the slower-acting P2Y receptor family during seizures. This includes data demonstrating expression changes of P2Y receptors in the hippocampus following acute seizures and during epilepsy and anticonvulsive properties of P2Y-targeting drugs; in particular drugs targeting the P2Y1 subtype. Seizures, however, also involve damage to extra-hippocampal brain regions such as the cortex, which is thought to contribute to the epileptic phenotype. To analyze expressional changes of the P2Y receptor family in the cortex following status epilepticus and to determine the impact of drugs interfering with P2Y1 signaling on cortical damage, we used a unilateral mouse model of intraamygdala kainic acid-induced status epilepticus. Analysis of cortical tissue showed that status epilepticus leads to a global up-regulation of the P2Y receptor family in the cortex including P2Y1, P2Y2, P2Y4, and P2Y6, with the P2Y1 and P2Y4 receptor subtypes showing the strongest increase. Supporting a detrimental role of P2Y1 activation during status epilepticus, treatment with the P2Y1 agonist MRS2365 exacerbated high frequency high amplitude spiking, synonymous with injury-causing electrographic activity, and treatment with the P2Y1 antagonists MRS2500 protected against seizure-induced cortical damage. Suggesting P2Y1-mediated effects are predominantly due to increased microglia activation, treatment with the broad-spectrum anti-inflammatory drug minocycline abolished the observed neuroprotective effects of P2Y1 antagonism. In conclusion, our results further support a role for P2Y1-mediated signaling during seizure generation and seizure-induced neurodegeneration, suggesting P2Y1-targeting therapies as novel treatment for drug-refractory status epilepticus.

Association of a P2RX7 gene missense variant with brachycephalic dog breeds.

Missense variants are associated with various phenotypic traits and disorders in dogs. The canine P2RX7 gene, coding the ATP-gated P2X7 receptor ion channel, contains four known missense variants. The current study aimed to examine the presence of these variants in a random sample of pedigree and mixed-pedigree dogs. Exons 3, 8, 11 and 13 of the P2RX7 gene, encoding these four respective variants, in 65 dogs were assessed by Sanger sequencing and combined with existing sequencing data from another 69 dogs. The distribution of these variants was then evaluated in all 134 dogs combined and separately within individual breeds including 35 different pure breeds. The rs23314713 (p.Phe103Leu) and rs23315462 (p.Pro452Ser) variants were present in 47 and 40% of all dogs studied respectively, with the rs23314713 variant associated with brachycephalic breeds. Among pedigree dogs, the rs23314713 and rs23315462 variants were associated with brachycephalic and non-brachycephalic breeds respectively. The rs851148233 (p.Arg270Cys) and rs850760787 (p.Arg365Gln) variants were present only in dogs of Cocker Spaniel and Labrador Retriever pedigrees respectively. No other missense variants were found in exons 3, 8, 11 and 13 of the P2RX7 gene within the dogs. In conclusion, the rs23314713 and rs23315462 missense variants of the P2RX7 gene are present in a large proportion of dogs, with the rs23314713 variant associated with a number of brachycephalic breeds. However, the association of this variant with dogs of bulldog ancestry, not brachycephaly per se, cannot be excluded.

Controlling Engineered P2X Receptors with Light.

This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors

The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration–response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC50 values of 0.76 μM, 22 μM, 763 μM or 118 μM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 μM ATA did not compete with [3H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration–response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αβ-methylene-ATP-sensitive P2X1 and P2X3 receptors.

P2X receptors mediated abnormal interaction between satellite glial cells and neurons in visceral pathological changes.

The adenosine triphosphate (ATP)-gated P2X receptor cation channel family consists of permeable ligand-gated ion channels that expand on the binding of extracellular adenosine 5'-ATP. ATP-gated P2X receptors are trimer ion channels that assemble homo or isomer from seven cloned subunits. P2X receptors are discovered mostly in mammalian and are being found in an increasing number of non-vertebrates, such as zebrafish, bullfrog, and ameba. P2X receptors are involved in many physiological processes, including regulation of heart rhythm and contractility, and regulation of pain, especially chronic pain and glia integration. This review summarizes the current studies on the regulation of P2X receptors in abnormal neuronal-glial interaction and the pathological changes in viscera, especially in myocardial ischemia.