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SignificanceATP is an important extracellular signal that activates P2X receptor channels. Although a large fraction of ATP is bound to divalent cations in vivo, the forms of ATP that activate P2X receptors are unknown. Here we show how the activity of homomeric P2X receptors is tuned by Mg2+ in some subtypes by preventing activation by free ATP, and in others by binding to a distinct regulatory site. We also find that both regulatory mechanisms are disengaged in heteromeric channels to form a sensitive ATP signaling pathway. These fundamental properties of P2X receptors will be valuable for investigating their physiological functions. The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.

Non-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α 1 -adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α 1 -adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)–induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84–124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12–24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,β-methylene-ATP (1.8–12.4%), 3.9% for β,γ-methylene-ATP (2.0–5.4%), 3.1% for 2-methylthio-ATP (− 0.1–6.3%), and 5.1% for ATPγS (1.0–9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.

At the neuromuscular junction, ATP and its metabolite adenosine regulate the release of acetylcholine by activating purinergic receptors (P2), thereby regulating the function of skeletal muscles under normal and pathological conditions. The presence of P2 receptors in the neuromuscular junction was assessed immunohistochemically on sections of m. soleus , m. extensor digitorum longus , and diaphragm muscle. Of the ionotropic P2X receptors, P2X1 and P2X2 subtypes showed high expression in the studied samples. Among the P2Y receptor family, only the P2Y13 subtype was expressed in the sections. This study provides new insight into the subtypes of purinergic receptors involved in controlling the cholinergic transmission in the mammalian neuromuscular system

Extracellular nucleotides have been recognized as important mediators of activation, triggering multiple responses via plasma membrane receptors known as P2 receptors. P2 receptors comprise P2X ionotropic receptors and G protein-coupled P2Y receptors. P2X receptors are expressed in many tissues, where they are involved in a number of functions including synaptic transmission, muscle contraction, platelet aggregation, inflammation, macrophage activation, differentiation and proliferation, neuropathic and inflammatory pain. P2X4 is one of the most sensitive purinergic receptors (at nanomolar ATP concentrations), about one thousand times more than the archetypal P2X7. P2X4 is widely expressed in central and peripheral neurons, in microglia, and also found in various epithelial tissues and endothelial cells. It localizes on the plasma membrane, but also in intracellular compartments. P2X4 is preferentially localized in lysosomes, where it is protected from proteolysis by its glycosylation. High ATP concentration in the lysosomes does not activate P2X4 at low pH; P2X4 gets activated by intra-lysosomal ATP only in its fully dissociated tetra-anionic form, when the pH increases to 7.4. Thus, P2X4 is functioning as a Ca -channel after the fusion of late endosomes and lysosomes. P2X4 modulates major neurotransmitter systems and regulates alcohol-induced responses in microglia. P2X4 is one of the key receptors mediating neuropathic pain. However, injury-induced upregulation of P2X4 expression is gender dependent and plays a key role in pain difference between males and females. P2X4 is also involved in inflammation. Extracellular ATP being a pro-inflammatory molecule, P2X4 can trigger inflammation in response to high ATP release. It is therefore involved in multiple pathologies, like post-ischemic inflammation, rheumatoid arthritis, airways inflammation in asthma, neurodegenerative diseases and even metabolic syndrome. Although P2X4 remains poorly characterized, more studies are needed as it is likely to be a potential therapeutic target in these multiple pathologies.

Shock wave treatment (SWT) is a non-invasive therapy applied in musculoskeletal and urological disorders, as well as in chronic wound regeneration. As the use of medical SWT broadens, it is important to better understand the molecular mechanisms underlying its success. Here, we identified P2X4 and P2Y2 purinergic receptors to be primarily expressed in C3H/10T1/2 mouse mesenchymal stromal cells and investigated their role in the initiation of the signaling events following SWT using single- and double-receptor knock-out (KO) cell lines. We show that SWT induced the expression of c-Jun and c-Fos within 30 min after stimulation and that the SWT-induced Erk1/2 pathway activation and immediate early gene expression were decreased in P2Y2-, P2X4- and P2Y2/P2X4-deficient cells. Importantly, SWT did not promote proliferation in P2Y2/P2X4-deficient cells, while loss of either one of the receptors significantly reduced the proliferative effect, indicating a cumulative effect of their loss. Finally, our data suggests a more prominent role of the P2Y2 receptor in SWT-induced cellular effects, since primarily its loss contributed to the observed changes. With these findings, we further the understanding of the molecular mechanisms of SWT and propose that the varying expression of purinergic receptors in tissues should be considered when establishing treatment protocols.

Nodose ganglion (NG) neurons are visceral primary sensory neurons. The transmission and regulation of visceral sensation is mediated mainly by the P2X purinoceptor (P2X receptor). Although the characteristics of different P2X receptor subunits in the NG have been studied previously, comprehensive analyses have not been performed. In this study, we used immunohistochemistry, immunocytochemistry, and whole cell patch clamp techniques to compare the expression and function of P2X1, P2X2, P2X3, and P2X4 receptor subunits in adult rat NG neurons. Polyclonal antibodies against the four P2X subunits labeled different subpopulations of NG neurons. P2X1 and P2X3 were expressed mainly in small-to-medium sized NG neurons, whereas P2X2 and P2X4 were located mostly in medium- and larger-sized NG neurons. Over 36% of NG neurons were P2X3 positive, which was higher than the other three P2X subunits. In addition, different types of currents were recorded from neurons expressing different P2X subunits. The fast type of ATP current was recorded from neurons containing P2X1-4 subunits, the intermediate type of current was recorded from neurons containing the P2X1, P2X3, and P2X4 subunits, the slow type was recorded from neurons expressing P2X1-3, and/or P2X4 subunits, whereas the very slow type was recorded from neurons containing the P2X2 and P2X3 subunits. These comparative results provide an anatomical verification of the different subunits in NG neurons, and offer direct support for the idea that various functional NG populations have distinct responses to ATP, which might be in part due to the different expression profiles of diverse P2X subunits.

Washing of platelets is an important procedure commonly used for experimental studies, e.g. in cardiovascular research. As a known phenomenon, responsiveness to adenosine diphosphate (ADP) is reduced in washed platelets, although underlying molecular mechanisms-potentially interfering with experimental results-have not been thoroughly studied. Since ADP mediates its effects via three purinergic receptors P2Y1, P2X1 and P2Y12, their surface expression and function were investigated in washed platelets and, for comparison, in platelet-rich-plasma (PRP) at different time points for up to 2 hours after preparation. In contrast to PRP, flow cytometric analysis of surface expression in washed platelets revealed an increase of all receptors during the first 60 minutes after preparation followed by a significant reduction, which points to an initial preactivation of platelets and consecutive degeneration. The activity of the P2X1 receptor (measured by selectively induced calcium flux) was substantially maintained in both PRP and washed platelets. P2Y12 function (determined by flow cytometry as platelet reactivity index) was partially reduced after platelet washing compared to PRP, but remained stable in course of ongoing storage. However, the function of the P2Y1 receptor (measured by selectively induced calcium flux) continuously declined after preparation of washed platelets. In conclusion, decreasing ADP responsiveness in washed platelets is particularly caused by impaired activity of the P2Y1 receptor associated with disturbed calcium regulation, which has to be considered in the design of experimental studies addressing ADP mediated platelet function.

Phosphoinositides modulate the function of several ion channels, including most ATP-gated P2X receptor channels in neurons and glia, but little is known about the underlying molecular mechanism. We identified a phosphoinositide-binding motif formed of two clusters of positively charged amino acids located on the P2X cytosolic C-terminal domain, proximal to the second transmembrane domain. For all known P2X subtypes, the specific arrangement of basic residues in these semi-conserved clusters determines their sensitivity to membrane phospholipids. Neutralization of these positive charges disrupts the functional properties of the prototypical phosphoinositide-binding P2X4 subtype, mimicking wortmannin-induced phosphoinositide depletion, whereas adding basic residues at homologous positions to the natively insensitive P2X5 subtype establishes de novo phosphoinositide-mediated regulation. Moreover, biochemical evidence of in vitro P2X subunit-phospholipid interaction and functional intracellular phosphoinositide-binding assays demonstrate that the dual polybasic cluster is necessary and sufficient for regulation of P2X signaling by phospholipids.

An X-ray structure of human P2Y 12 receptor, a clinical drug target for platelet aggregation inhibitors, is presented in complex with an agonist, providing insight into the δ-group of class A G-protein-coupled receptors. Key platelet aggregation GPCR structures Two papers in this issue of Nature present the crystal structures of the human P2Y 12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y 12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y 12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. The P2Y 12 receptor (P2Y 12 R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y 12 R provided useful insights into ligand binding 1 , 2 , 3 , 4 , the agonist and antagonist recognition and function at the P2Y 12 R remain poorly understood at the molecular level. Here we report the structures of the human P2Y 12 R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. These structures, together with the structure of the P2Y 12 R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283) 5 , reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y 12 R, with only partially overlapped binding pockets. The agonist-bound P2Y 12 R structure answers long-standing questions surrounding P2Y 12 R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y 12 R and potentially for other closely related P2YRs.

Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1-6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell (EC)-specific Lpa4;Lpa6 DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased β-catenin- and Notch intracellular domain (NICD)-mediated endothelial expression of the Notch ligand delta-like 4 (DLL4). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Of note, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6 DKO mice. Overall, these results suggest that the Gα12/Gα13-coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide a novel insight into the biology of GPCR-activated YAP/TAZ.