Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 3: Structure-activity relationships of pyrropyrazolone derivatives.

The P2X3 receptor is an attractive target for the treatment of pain and chronic coughing, and thus P2X3 antagonists have been developed as new therapeutic drugs. We previously reported selective P2X3 receptor antagonists by derivatization of hit compound 1. As a result, we identified hit compound 3, the structure of which was similar to hit compound 1. On the basis of SAR studies of hit compound 1, we modified hit compound 3 and compound 42 was identified as having analgesic efficacy by oral administration.

Spatiotemporal identification of druggable binding sites using deep learning.

Identification of novel protein binding sites expands druggable genome and opens new opportunities for drug discovery. Generally, presence or absence of a binding site depends on the three-dimensional conformation of a protein, making binding site identification resemble the object detection problem in computer vision. Here we introduce a computational approach for the large-scale detection of protein binding sites, that considers protein conformations as 3D-images, binding sites as objects on these images to detect, and conformational ensembles of proteins as 3D-videos to analyze. BiteNet is suitable for spatiotemporal detection of hard-to-spot allosteric binding sites, as we showed for conformation-specific binding site of the epidermal growth factor receptor, oligomer-specific binding site of the ion channel, and binding site in G protein-coupled receptor. BiteNet outperforms state-of-the-art methods both in terms of accuracy and speed, taking about 1.5 minutes to analyze 1000 conformations of a protein with ~2000 atoms.

Exploring the molecular mechanism of the effect of puerarin on P2X3.

P2X3 is a ligand-gated nonselective cation channel and permeable to Na+, K+ and Ca2+. Adenosine triphosphate (ATP) activation of the P2X3 on primary sensory ganglion neurons is involved in nociceptive transmission. Puerarin is a major active ingredient extracted from the traditional Chinese medicine Ge-gen. Puerarin inhibits nociceptive signal transmission by inhibiting the P2X3 in the dorsal root ganglia (DRG) and sympathetic ganglia, but its molecular mechanism is unclear. The aim of this study was to explore the molecular mechanism of puerarin on the P2X3. Here, molecular docking results revealed that puerarin binds well to the human P2X3 protein in the vicinity of the ATP binding pocket. Protein-ligand docking showed that the V64A mutation reduced the effect of puerarin but had little effect on ATP. V64A site-directed mutagenesis of P2X3 was performed using an overlap extension PCR technique. The wild-type and V64A mutant pEGFP-C1-P2X3 recombinant plasmids were transfected into HEK 293 cells. The electrophysiology results demonstrated that puerarin exerted an obvious inhibitory effect on ATP-activated currents in HEK 293 cells transfected with the wild-type P2X3, while little inhibition was observed in HEK 293 cells transfected with the mutant P2X3. These studies suggest that puerarin inhibits the P2X3 by binding to V64A.

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.

Molecular mechanisms of human P2X3 receptor channel activation and modulation by divalent cation bound ATP.

P2X3 receptor channels expressed in sensory neurons are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization, making them attractive therapeutic targets. Although several P2X3 structures are known, it is unclear how physiologically abundant Ca2+-ATP and Mg2+-ATP activate the receptor, or how divalent cations regulate channel function. We used structural, computational and functional approaches to show that a crucial acidic chamber near the nucleotide-binding pocket in human P2X3 receptors accommodates divalent ions in two distinct modes in the absence and presence of nucleotide. The unusual engagement between the receptor, divalent ion and the γ-phosphate of ATP enables channel activation by ATP-divalent complex, cooperatively stabilizes the nucleotide on the receptor to slow ATP unbinding and recovery from desensitization, a key mechanism for limiting channel activity. These findings reveal how P2X3 receptors recognize and are activated by divalent-bound ATP, aiding future physiological investigations and drug development.

Druggable negative allosteric site of P2X3 receptors.

Allosteric modulation provides exciting opportunities for drug discovery of enzymes, ion channels, and G protein-coupled receptors. As cation channels gated by extracellular ATP, P2X receptors have attracted wide attention as new drug targets. Although small molecules targeting P2X receptors have entered into clinical trials for rheumatoid arthritis, cough, and pain, negative allosteric modulation of these receptors remains largely unexplored. Here, combining X-ray crystallography, computational modeling, and functional studies of channel mutants, we identified a negative allosteric site on P2X3 receptors, fostered by the left flipper (LF), lower body (LB), and dorsal fin (DF) domains. Using two structurally analogous subtype-specific allosteric inhibitors of P2X3, AF-353 and AF-219, the latter being a drug candidate under phase II clinical trials for refractory chronic cough and idiopathic pulmonary fibrosis, we defined the molecular interactions between the drugs and receptors and the mechanism by which allosteric changes in the LF, DF, and LB domains modulate ATP activation of P2X3. Our detailed characterization of this druggable allosteric site should inspire new strategies to develop P2X3-specific allosteric modulators for clinical use.

PyContact: Rapid, Customizable, and Visual Analysis of Noncovalent Interactions in MD Simulations.

Molecular dynamics (MD) simulations have become ubiquitous in all areas of life sciences. The size and model complexity of MD simulations are rapidly growing along with increasing computing power and improved algorithms. This growth has led to the production of a large amount of simulation data that need to be filtered for relevant information to address specific biomedical and biochemical questions. One of the most relevant molecular properties that can be investigated by all-atom MD simulations is the time-dependent evolution of the complex noncovalent interaction networks governing such fundamental aspects as molecular recognition, binding strength, and mechanical and structural stability. Extracting, evaluating, and visualizing noncovalent interactions is a key task in the daily work of structural biologists. We have developed PyContact, an easy-to-use, highly flexible, and intuitive graphical user interface-based application, designed to provide a toolkit to investigate biomolecular interactions in MD trajectories. PyContact is designed to facilitate this task by enabling identification of relevant noncovalent interactions in a comprehensible manner. The implementation of PyContact as a standalone application enables rapid analysis and data visualization without any additional programming requirements, and also preserves full in-program customization and extension capabilities for advanced users. The statistical analysis representation is interactively combined with full mapping of the results on the molecular system through the synergistic connection between PyContact and VMD. We showcase the capabilities and scientific significance of PyContact by analyzing and visualizing in great detail the noncovalent interactions underlying the ion permeation pathway of the human P2X3 receptor. As a second application, we examine the protein-protein interaction network of the mechanically ultrastable cohesin-dockering complex.

Purinergic and adenosine receptors contribute to hypoxic hyperventilation in zebrafish (Danio rerio).

The chemoreceptors involved in oxygen sensing in teleost fish are neuroepithelial cells (NECs) in the gills, and are analogous to glomus cells in the mammalian carotid body. Purinergic signalling mechanisms involving the neurotransmitters, ATP and adenosine, have been identified in mediating hypoxic signalling in the carotid body, but these pathways are not well understood in the fish gill. The present study used a behavioural assay to screen for the effects of drugs, that target purinergic and adenosine receptors, on the hyperventilatory response to hypoxia in larval zebrafish (Danio rerio) in order to determine if the receptors on which these drugs act may be involved in hypoxic signalling. The purinergic receptor antagonist, PPADS, targets purinergic P2X2/3 receptors and inhibited the hyperventilatory response to hypoxia (IC50=18.9µM). The broad-spectrum purinergic agonist, ATPγS, elicited a hyperventilatory response (EC50=168µM). The non-specific adenosine receptor antagonist, caffeine, inhibited the hyperventilatory response to hypoxia, as did the specific A2a receptor antagonist, SCH58261 (IC50=220nM). These results suggest that P2X2/3 and A2a receptors are candidates for mediating hypoxic hyperventilation in zebrafish. This study highlights the potential of applying chemical screening to ventilatory behaviour in zebrafish to further our understanding of the pathways involved in signalling by gill NECs and oxygen sensing in vertebrates.

X-ray structures define human P2X(3) receptor gating cycle and antagonist action.

P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the 'cytoplasmic cap', which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, meaning that the human P2X7R (hP2X7R) is an attractive therapeutic target. The crystal structures of the zebrafish P2X4R in the closed and ATP-bound open states provide an unprecedented opportunity for structure-guided identification of new ligands. The present study performed virtual screening of ∼100,000 structurally diverse compounds against the ATP-binding pocket in the hP2X7R. This identified three compounds (C23, C40 and C60) out of 73 top-ranked compounds by testing against hP2X7R-mediated Ca(2+) responses. These compounds were further characterised using Ca(2+) imaging, patch-clamp current recording, YO-PRO-1 uptake and propidium iodide cell death assays. All three compounds inhibited BzATP-induced Ca(2+) responses concentration-dependently with IC50s of 5.1±0.3µM, 4.8±0.8µM and 3.2±0.2µM, respectively. C23 and C40 inhibited BzATP-induced currents in a reversible and concentration-dependent manner, with IC50s of 0.35±0.3µM and 1.2±0.1µM, respectively, but surprisingly C60 did not affect BzATP-induced currents up to 100µM. They suppressed BzATP-induced YO-PRO-1 uptake with IC50s of 1.8±0.9µM, 1.0±0.1µM and 0.8±0.2µM, respectively. Furthermore, these three compounds strongly protected against ATP-induced cell death. Among them, C40 and C60 exhibited strong specificity towards the hP2X7R over the hP2X4R and rP2X3R. In conclusion, our study reports the identification of three novel hP2X7R antagonists with micromolar potency for the first time using a structure-based approach, including the first P2X7R antagonist with preferential inhibition of large pore formation.

New P2X3 receptor antagonists. Part 2: Identification and SAR of quinazolinones.

Numerous potent P2X3 antagonists have been discovered and the therapeutic potential of P2X3 antagonism already comprises proof-of-concept data obtained in clinical trials with the most advanced compound. We have lately reported the discovery and optimization of thia-triaza-tricycle compounds with potent P2X3 antagonistic properties. This Letter describes the SAR of a back-up series containing a 4-oxo-quinazoline central ring. The discovery of the highly potent compounds 51 is presented.

New P2X3 receptor antagonists. Part 1: Discovery and optimization of tricyclic compounds.

Purinergic P2X3 receptors are trimeric ligand-gated ion channels whose antagonism is an appealing yet challenging and not fully validated drug development idea. With the aim of identification of an orally active, potent human P2X3 receptor antagonist compound that can penetrate the central nervous system, the compound collection of Gedeon Richter was screened. A hit series of tricyclic compounds was subjected to a rapid, two-step optimization process focusing on increasing potency, improving metabolic stability and CNS penetrability. Attempts resulted in compound 65, a potential tool compound for testing P2X3 inhibitory effects in vivo.

Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating.

P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify inter-subunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gating-induced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Charge-reversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.

Promoted Interaction of Nuclear Factor-κB With Demethylated Purinergic P2X3 Receptor Gene Contributes to Neuropathic Pain in Rats With Diabetes.

Painful diabetic neuropathy is a common complication of diabetes produced by mechanisms that as yet are incompletely defined. The aim of this study was to investigate the roles of nuclear factor-κB (NF-κB) in the regulation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3R) plasticity in dorsal root ganglion (DRG) neurons of rats with painful diabetes. Here, we showed that hindpaw pain hypersensitivity in streptozocin-induced diabetic rats was attenuated by treatment with purinergic receptor antagonist suramin or A-317491. The expression and function of P2X3Rs was markedly enhanced in hindpaw-innervated DRG neurons in diabetic rats. The CpG (cytosine guanine dinucleotide) island in the p2x3r gene promoter region was significantly demethylated, and the expression of DNA methyltransferase 3b was remarkably downregulated in DRGs in diabetic rats. The binding ability of p65 (an active form of NF-κB) with the p2x3r gene promoter region and p65 expression were enhanced significantly in diabetes. The inhibition of p65 signaling using the NF-κB inhibitor pyrrolidine dithiocarbamate or recombinant lentiviral vectors designated as lentiviral vector-p65 small interfering RNA remarkably suppressed P2X3R activities and attenuated diabetic pain hypersensitivity. Insulin treatment significantly attenuated pain hypersensitivity and suppressed the expression of p65 and P2X3Rs. Our findings suggest that the p2x3r gene promoter DNA demethylation and enhanced interaction with p65 contributes to P2X3R sensitization and diabetic pain hypersensitivity.

Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening.

BACKGROUND AND PURPOSE: It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACH: A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTS: A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,ß-methylene ATP (α,ß-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,ß-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONS: In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Pyrid-2-yl and 2-CyanoPhenyl fused heterocyclic compounds as human P2X3 inhibitors: a combined approach based on homology modelling, docking and QSAR analysis.

P2X receptors are hetero-oligomeric proteins that function as membrane ion channels and are gated by extracellular ATP. The hP2X[Formula: see text] subunit is a constituent of the channels on a subset of sensory neurons involved in pain signaling, where ATP released by damaged and inflamed tissue can initiate action potentials. Hence, the inhibition of ATP-activated P2X3 receptor is an exciting approach for the treatment of inflammatory and neuropathic pain. Recently, the crystal structures of zebrafish P2X4 (zP2X4) were obtained in closed, apo state (PDB ID: 3I5D) and ATP-bound, open state (PDB ID: 4DW1). These structures were used to develop a homology model of human P2X3 (hP2X3 in order to identify through docking studies, the binding modes of known P2X3 inhibitors and their key active site interactions, along with a pharmacophore-based 3D-QSAR model for a series of 136 Pyrid-2-yl and 2-CyanoPhenyl fused heterocyclic compounds. These 3D-QSAR models have been developed with different combinations of training and test set divisions obtained by random separation, Jarvis-Patrick clustering, K-means clustering and sphere exclusion methods. The best predictive 3D-QSAR model resulted in training set R2 of 0.75, internal test set Q2 of 0.74, Pearson-R value of 0.87 and root mean square error of 0.37. The information generated by the pharmacophore model and docking analyses using the homology model provides valuable clues to design novel potent hP2X3 inhibitors.

Purinergic autocrine regulation of mechanosensitivity and serotonin release in a human EC model: ATP-gated P2X3 channels in EC are downregulated in ulcerative colitis.

BACKGROUND: Alterations in 5-hydroxytryptamine (HT) signaling in inflamed gut may contribute to pathogenesis of inflammatory bowel diseases. Adenosine 5'-triphosphate (ATP) regulates mucosal-mechanosensory reflexes and ATP receptors are sensitive to mucosal inflammation. Yet, it remains unknown whether ATP can modulate 5-HT signaling in enterochromaffin cells (EC). We tested the novel purinergic hypothesis that ATP is a critical autocrine regulator of EC mechanosensitivity and whether EC expression of ATP-gated P2X3-ion channels is altered in inflammatory bowel diseases. METHODS: Laser confocal (fluo-4) Ca imaging was performed in 1947 BON cells. Chemical stimulation or mechanical stimulation (MS) was used to study 5-HT or ATP release in human BON or surgical mucosal specimens, and purine receptors by reverse transcription-polymerase chain reaction, Western Blot, or P2X3-immunoreactivity in BON or 5-HT human EC (hEC) in 11 control and 10 severely inflamed ulcerative colitis (UC) cases. RESULTS: ATP or MS triggered Ca-transients or 5-HT release in BON. ATP or adenosine diphosphate increased 5-HT release 5-fold. MS caused ATP release, detected after 5'ecto-ATPase inhibition by ARL67156. ARL67156 augmented and apyrase blocked Ca/5-HT mechanosensitive responses. 2-Methyl-thio-adenosine diphosphate 5'-monophosphate-evoked (P2Y1,12) or mechanically-evoked responses were blocked or augmented by a P2Y1,12 antagonist, MRS2179, in different cells or inhibited by U73122. A P2Y12 antagonist, 2MeSAMP, augmented responses. A P2X1,3 agonist, α,ß-MeATP, triggered Ca responses, whereas a P2X1,2/3,3 antagonist, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, blocked mechanical responses or cell-surface 5'ATP- labeling. In hEC, α,ß-MeATP stimulated 5-HT release. In UC, P2X3-immunoreactivity decreased from 15% to 0.2% of 5-HThECs. Human mucosa and BON expressed P2X1, P2X3, P2X4, P2X5, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12R-messenger RNA transcripts. CONCLUSIONS: ATP is a critical determinant of mechanosensation and 5-HT release via autocrine activation of slow P2Y1-phospholipase C/inositol-1,4,5-triphosphate-Ca or inhibitory P2Y12-purinergic pathways, and fast ATP-gated P2X3-channels. UC downregulation of P2X3-channels (or A2B) is postulated to mediate abnormal 5-HT signaling.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+.

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 Mg(2+). Here we investigated the active forms of ATP and found that the action of MgATP(2-) and ATP(4-) differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP(2-) promotes opening with very low efficacy. In contrast, both free ATP and MgATP(2-) robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg(2+) 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 MgATP(2-) and weak regulation by Mg(2+). These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP(2-) and regulation by Mg(2+), and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.