ATP-gated Non-selective Cation Channels Research Articles

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.

Spinal P2X4 Receptors Involved in Visceral Hypersensitivity of Neonatal Maternal Separation Rats.

Recent studies have demonstrated the vital role of P2X4 receptors (a family of ATP-gated non-selective cation channels) in the transmission of neuropathic and inflammatory pain. In this study, we investigated the role of spinal P2X4 receptors in chronic functional visceral hypersensitivity of neonatal maternal separation (NMS) rats. A rat model of irritable bowel syndrome was established by neonatal maternal separation. Visceral sensitivity was assessed by recording the response of the external oblique abdominal muscle to colorectal distension. P2X4 receptor antagonist and agonist were administrated intrathecally. The expression of P2X4 receptor was examined by Western Blot and immunofluorescence. The effect of P2X4 receptor antagonist on expression of brain-derived neurotrophic factor (BDNF) was assessed by Western Blot. We found neonatal maternal separation enhanced visceral hypersensitivity and increased the expression of P2X4 receptor in spinal thoracolumbar and lumbosacral segments of rats. Pharmacological results showed that visceral sensitivity was attenuated after intrathecal injection of P2X4 receptor antagonist, 5-BDBD, at doses of 10nM or 100nM, while visceral sensitivity was enhanced after intrathecal injection of P2X4 receptor agonist C5-TDS at doses of 10μM or 15μM. In addition, the spinal expression of BDNF significantly increased in NMS rats and intrathecal injection of 5-BDBD significantly decreased the expression of BDNF especially in NMS rats. C5-TDS failed to increase EMG amplitude in the presence of ANA-12 in control rats. Our results suggested the spinal P2X4 receptors played an important role in visceral hypersensitivity of NMS rats through BDNF.

Purinergic Signaling in Spermatogenesis.

Adenosine triphosphate (ATP) serves as the essential source of cellular energy. Over the last two decades, however, ATP has also attracted increasing interest as an extracellular signal that activates purinergic plasma membrane receptors of the P2 family. P2 receptors are divided into two types: ATP-gated nonselective cation channels (P2X) and G protein-coupled receptors (P2Y), the latter being activated by a broad range of purine and pyrimidine nucleotides (ATP, ADP, UTP, and UDP, among others). Purinergic signaling mechanisms are involved in numerous physiological events and pathophysiological conditions. Here, we address the growing body of evidence implicating purinergic signaling in male reproductive system functions. The life-long generation of fertile male germ cells is a highly complex, yet mechanistically poorly understood process. Given the relatively sparse innervation of the testis, spermatogenesis relies on both endocrine control and multi-directional paracrine communication. Therefore, a detailed understanding of such paracrine messengers, including ATP, is crucial to gain mechanistic insight into male reproduction.⁠

Role of TRPV4-P2X7 Pathway in Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation channel that is involved in the development of neuropathic pain. P2X7 receptor (P2X7) belongs to a class of ATP-gated nonselective cation channels that plays an important role in neuropathic pain. Nevertheless, little is known about the interaction between them for neuropathic pain. In this paper, we investigated role of TRPV4-P2X7 pathway in neuropathic pain. We evaluated the effect of TRPV4-P2X7 pathway on neuropathic pain in a chronic compression of the dorsal root ganglion (DRG) (hereafter termed CCD) model. We analyzed the effect of P2X7 on mechanical and thermal hyperalgesia mediated by TRPV4 in CCD. Furthermore, we assessed the effect of TRPV4 on the expression of P2X7 and the release of IL-1β and IL-6 in DRG after CCD. We found that intraperitoneal injection of TRPV4 agonist GSK-1016790A led to a significant increase of mechanical and thermal hyperalgesia in CCD, which was partially suppressed by P2X7 blockade with antagonist Brilliant Blue G (BBG). Then, we further noticed that GSK-1016790A injection increased the P2X7 expression of CCD, which was decreased by TRPV4 blockade with antagonist RN-1734 and HC-067047. Furthermore, we also discovered that the expressions of IL-1β and IL-6 were upregulated by GSK-1016790A injection but reduced by RN-1734 and HC-067047. Our results provide evidence that P2X7 contributes to development of neuropathic pain mediated by TRPV4 in the CCD model, which may be the basis for treatment of neuropathic pain relief.

New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1β Release: An Involvement in its Alleviating Effect on Neuropathic Pain.

P2X7 receptors (P2X7Rs) belong to a family of ATP-gated non-selective cation channels. Microglia represent a major cell type expressing P2X7Rs. The activation of microglial P2X7Rs causes the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β). This response has been implicated in neuroinflammatory states in the central nervous system and in various diseases, including neuropathic pain. Thus, P2X7R may represent a potential therapeutic target. In the present study, we screened a chemical library of clinically approved drugs (1979 compounds) by high-throughput screening and showed that the Ca2+ channel blocker cilnidipine has an inhibitory effect on rodent and human P2X7R. In primary cultured rat microglial cells, cilnidipine inhibited P2X7R-mediated Ca2+ responses and IL-1β release. Moreover, in a rat model of neuropathic pain, the intrathecal administration of cilnidipine produced a reversal of nerve injury-induced mechanical hypersensitivity, a cardinal symptom of neuropathic pain. These results point to a new inhibitory effect of cilnidipine on microglial P2X7R-mediated inflammatory responses and neuropathic pain, proposing its therapeutic potential.

P2X7 receptor: a critical regulator and potential target for breast cancer.

Breast cancer is currently the most common cancer and the leading cause of cancer death among women worldwide. Advanced breast cancer is prone to metastasis, and there is currently no drug to cure metastatic breast cancer. The purinergic ligand-gated ion channel 7 receptor is an ATP-gated nonselective cation channel receptor and is involved in signal transduction, growth regulation, cytokine secretion, and tumor cell development. Recent studies have shown that upregulation of the P2X7 receptor in breast cancer can mediate AKT signaling pathways, Ca2 þ-activated SK3 potassium channels, and EMT and regulate the secretion of small extracellular vesicles to promote breast cancer invasion and migration, which are affected by factors such as hypoxia and ATP. In addition, studies have shown that microRNAs can bind to the 3' untranslated region of the P2X7 receptor, which affects the occurrence and development of breast cancer by upregulating and downregulating P2X7 receptor expression. Studies have shown that new P2X7 receptor inhibitors, such as emodin and Uncaria tomentosa, can inhibit P2X7 receptor-mediated breast cancer invasion and are expected to be used clinically. This article reviews the research progress on the relationship between the P2X7 receptor and breast cancer to provide new ideas and a basis for clinical diagnosis and treatment.

Transient receptor potential ankyrin 1 contributes to the ATP-elicited oxidative stress and inflammation in THP-1-derived macrophage.

P2X7 receptor (P2X7R) is an ATP-gated non-selective cation channel which mediates ATP-induced inflammation in macrophages. Transient receptor potential (TRP) receptors are nociceptors in cellular membrane which can perceive the stimuli of environmental irritant. The interaction between TRP channels and P2X7R has been found while the details about inflammation are still unclear. In this study, we suggested that transient receptor potential ankyrin 1 (TRPA1), a member of TRP superfamily, participates in ATP-induced oxidative stress and inflammation in human acute monocytic leukemia cell line (THP-1)-derived macrophage. The co-localization between TRPA1 and P2X7R was detected using immunofluorescence in THP-1-derived macrophage and transfected human embryonic kidney cell line (HEK293T). The mechanism by which ATP or 3'-O-(4-Benzoylbenzoyl)-ATP (BzATP) induces the activation of macrophages was verified by calcium imaging, mitochondrial reactive oxygen species (mtROS) detection, mitochondrial membrane potential (∆Ψm) measurement, flow cytometry, enzyme-linked immunosorbent assay (ELISA), western blotting, CCK-8 assay, and the lactate dehydrogenase (LDH) release cytotoxic assay. The BzATP and ATP induced calcium overload, mitochondria injury, interleukin-1β (IL-1β) secretion, and cytotoxicity can be inhibited by TRPA1 antagonists. These results indicated that TRPA1 can co-localize with P2X7R and mediate ATP-induced oxidative stress and inflammation. Therefore, the inhibition of TRPA1 may provide a potential therapy for ATP-elicited inflammatory diseases, including atherosclerosis.

Green Pharma: A New Strategy for Drug Discovery in Academia by Targeting Glial Cells and ATP Receptors

Neuropathic pain associated with cancer, diabetic neuropathy, and postherpetic neuralgia is a type of intractable chronic pain characterized by mechanical allodynia and abnormal pain hypersensitivity evoked by innocuous stimuli. However, this disorder has no specific treatment. We previously showed that the purinergic receptor P2X4 (P2X4R), a subtype of ATP-gated nonselective cation channels, is highly upregulated in spinal microglia after peripheral nerve injury, and blocking the function of P2X4R reverses mechanical allodynia. In the present study, we screened a chemical library of 1979 clinically approved compounds (a gift from the Drug Discovery Initiative at the University of Tokyo) aimed at achieving "Eco-Pharma," which refers to seeking new effects of existing drugs. We demonstrated that duloxetine, a serotonin and noradrenaline reuptake inhibitor, has an inhibitory effect on rat and human P2X4R. In rat primary cultured microglial cells, duloxetine also inhibited P2X4R-mediated responses. Moreover, intrathecal administration of duloxetine in a model of neuropathic pain reversed nerve injury-induced mechanical allodynia. Based on those results, we suggest that the inhibition of P2X4R expressed in microglial cells may be involved in the antiallodynic effect of duloxetine in neuropathic pain. Furthermore, in this review, we discuss a new strategy for drug discovery called "Green Pharma" (a merger of "Eco-Pharma" and "Green chemistry" and referring to the development of eco-friendly pharmaceuticals).

P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1β and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1β, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1β release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1β release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.

Duloxetine Inhibits Microglial P2X4 Receptor Function and Alleviates Neuropathic Pain after Peripheral Nerve Injury.

P2X4 receptors (P2X4R) are a family of ATP-gated non-selective cation channels. We previously demonstrated that activation of P2X4R in spinal microglia is crucial for neuropathic pain, a highly debilitating chronic pain condition, suggesting that P2X4R is a potential therapeutic target for treating neuropathic pain. Thus, the identification of a compound that has a potent inhibitory effect on P2X4R is an important clinical challenge. In the present study, we screened a chemical library of clinically approved drugs and show for the first time that duloxetine, a serotonin and noradrenaline reuptake inhibitor, has an inhibitory effect on rodent and human P2X4R. In primary cultured microglial cells, duloxetine also inhibited P2X4R-, but not P2X7R-, mediated responses. Moreover, intrathecal administration of duloxetine in a model of neuropathic pain produced a reversal of nerve injury-induced mechanical allodynia, a cardinal symptom of neuropathic pain. In rats that were pretreated with a serotonin-depleting agent and a noradrenaline neurotoxin, the antiallodynic effect of duloxetine was reduced, but still remained. Based on these results, we suggest that, in addition to duloxetine’s primary inhibitory action on serotonin and noradrenaline transporters, an inhibitory effect on P2X4R may be involved at least in part in an antiallodynic effect of intrathecal duloxetine in a model of neuropathic pain.

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

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

Exploring the ATP-binding site of P2X receptors

P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation, and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors.

The Gating Mechanism of a P2X4 Receptor: Normal Mode Analysis and Molecular Dynamics Simulations

P2X4 receptors are trimeric ATP-gated non-selective cation channels which play crucial roles in various physiological processes. It remains unclear how ATP binding triggers channel opening. Here, we propose a gating mechanism of a P2X4 receptor based on normal mode analysis and molecular dynamics (MD) simulations. Starting with the first available P2X4 crystal structure in the resting state, a normal mode that couples the motions of three β strands (β1, β13 and β14) at the trimeric interface of the ligand binding domain (LBD) and the motion of the pore-forming helix (TM2) of the transmembrane domain (TMD) was identified. The resulting widening of the fenestrations above the TMD and the opening of the TMD pore are in close agreement with observed signatures of channel activation. Four charged residues implicated in ATP binding are located in β1, β13 and β14. P2X4 activation was further investigated by MD simulations in explicit water. ATP was placed near the putative binding site in two opposite orientations, with the adenine either promixal or distal to the TMD. In simulations with proximal adenine, the adenine ring inserted between β1 and β13 and the phosphate group moved downward. At the same time β1 and β14 approached each other to close in on the ATP, allowing close interactions with the four charged residues. The motions of these β strands are similar to those in the normal mode putatively representing channel activation. In simulations with distal adenine, the ATP hindered the closure between β1 and β14, perhaps representing a desensitized state. Our computational studies produced the first complete model, supported by electrophysiological data, for how ATP binding leads to P2X4 channel activation. The detailed gating mechanism will be essential for the rational drug design.

The Physiology, Pharmacology and Future of P2X7 as An Analgesic Drug Target: Hype or Promise?

P2X7 is an ATP-gated non-selective cation channel expressed primarily on cells of hematopoietic origin, such as macrophages and microglia. Since the initial cloning of this channel, enormous progress has been made in the understanding of the physiology, pharmacology and therapeutic utility of P2X7. This article attempts to review the biology of P2X7 with a focus on the complex pharmacology of this channel. Finally, the authors discuss the role of P2X7 as an analgesic drug target and raise some of the challenges and issues that face the P2X7 research community.

Activation and Regulation of Purinergic P2X Receptor Channels

Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.

Allosteric modulation of ATP-gated P2X receptor channels.

Seven mammalian purinergic receptor subunits, denoted P2X1-P2X7, and several spliced forms of these subunits have been cloned. When heterologously expressed, these cDNAs encode ATP-gated non-selective cation channels organized as trimers. All activated receptors produce cell depolarization and promote Ca(2+) influx through their pores and indirectly by activating voltage-gated calcium channels. However, the biophysical and pharmacological properties of these receptors differ considerably, and the majority of these subunits are also capable of forming heterotrimers with other members of the P2X receptor family, which confers further different properties. These channels have three ATP binding domains, presumably located between neighboring subunits, and occupancy of at least two binding sites is needed for their activation. In addition to the orthosteric binding sites for ATP, these receptors have additional allosteric sites that modulate the agonist action at receptors, including sites for trace metals, protons, neurosteroids, reactive oxygen species and phosphoinositides. The allosteric regulation of P2X receptors is frequently receptor-specific and could be a useful tool to identify P2X members in native tissues and their roles in signaling. The focus of this review is on common and receptor-specific allosteric modulation of P2X receptors and the molecular base accounting for allosteric binding sites.

Genetic and Functional Analysis of Human P2X5 Reveals a Distinct Pattern of Exon 10 Polymorphism with Predominant Expression of the Nonfunctional Receptor Isoform

P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive correlation between genotype and P2X5 transcript. Immunostaining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiological analysis showed robust ATP-evoked currents when full-length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full-length isoform is known.

Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.

SummaryAcid-sensing ion channels are proton-activated, sodium-selective channels composed of three subunits and members of the superfamily of epithelial sodium channels, mechanosensitive and FMRF peptide-gated ion channels. These ubiquitous eukaryotic ion channels play essential roles in biological activities as diverse as sodium homeostasis, taste, and pain. Despite their crucial roles in biology and their unusual trimeric subunit stoichiometry, there is little knowledge of the structural and chemical principles underlying their ion channel architecture and ion binding sites. Here we describe the structure of a functional acid sensing ion channel in a desensitized state at 3 Å resolution, the location and composition of the ~8 Å ‘thick’ desensitization gate, and the trigonal antiprism coordination of cesium ions bound in the extracellular vestibule. Comparison of the acid sensing ion channel structure with the ATP-gated P2×4 receptor reveals similarity in pore architecture and aqueous vestibules, suggesting unanticipated yet common structural and mechanistic principles.

Crystal structure of the ATP-gated P2X4 ion channel in the closed state

SummaryP2X receptors are cation selective ion channels gated by extracellular ATP and implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is presently no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X4 receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in β-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. Within the transmembrane pore, the ‘gate’ is defined by an ~8 Ǻ slab of protein. We define the location of three non-canonical, intersubunit ATP binding sites and suggest that ATP binding promotes subunit rearrangement and ion channel opening.

Trimeric ion-channel design

Cavernous chambers, intricate passages, a gate with a curious lock — the structure of an ATP-activated ion channel reveals its architecture. And this intriguing interior design is found in another type of ion channel too. P2X receptors are ATP-gated non-selective cation channels involved in nociception and inflammatory responses, whose structures were unknown. Kawate et al. now present the crystal structure of the zebrafish P2X4 receptor in a closed state. The trimeric structure reveals some of the molecular underpinnings of ligand-binding, cation entry and channel gating. A related paper presents the structure of chicken acid-sensing ion channel 1 (ASIC1) in a desensitized state. Like P2X receptors, ASICs are trimeric, but they belong to an entirely different family of ion channels. The structure determination of ASIC1 shows how ion permeation and desensitization may occur, and comparison of ASIC and P2X structures suggests that these functionally distinct channels employ similar mechanistic principles.