Ethanol differentially affects ATP-gated P2X(3) and P2X(4) receptor subtypes expressed in Xenopus oocytes

P2X receptors are cation-selective, ligand-gated ion channels activated by synaptically released, extracellular adenosine 5'-triphosphate (ATP). ATP-gated currents are inhibited by ethanol when tested in dorsal root ganglion and CA1 neurons. Recently, we reported differences in sensitivity to ethanol inhibition between homomeric P2X(2) and P2X(4) receptors expressed in Xenopus oocytes, which suggested that subunit composition of native P2X receptors determines their ethanol sensitivity. The present study extended the investigation to P2X(3) receptors. The effects of ethanol and zinc ions (Zn(2+)) were tested on homomeric P2X(3) and P2X(4) receptors expressed in Xenopus oocytes using two-electrode voltage clamp. Ethanol potentiated ATP-gated P2X(3) receptor currents in a concentration dependent manner. In contrast, ethanol inhibited P2X(4) receptor function. Ethanol did not directly alter receptor function, nor did it alter the Hill coefficient or maximal ATP response (E(max)) in either P2X(3) or P2X(4) receptors. Ethanol increased the maximal response to Zn(2+) ATP-gated currents in P2X3 receptors which suggests that ethanol and Zn(2+) act on different sites. The differences in ethanol response of P2X(3) and P2X(4) receptors set the stage for future investigations that will use chimeric P2X receptors or other molecular manipulations of P2X structure to investigate the molecular sites and mechanisms of action of ethanol.     

Current perspectives on acid-sensing ion channels: new advances and therapeutic implications

Acid-sensing ion channels (ASICs) form a family of voltage-independent cation channels that predominantly conduct Na⁺ ions, and were identified at the molecular level a little more than a decade ago. ASICs are activated by extracellular acidification within the physiological range, and they form effective proton sensors in both central and peripheral sensory neurons. A combination of genetic and pharmacologic approaches has revealed their implication in an increasing number of physiological and pathophysiological processes – most of them associated with extracellular pH fluctuations, ranging from synaptic plasticity, learning, memory, fear, depression, seizure termination and neuronal degeneration to nociception and mechanosensation. ASICs, therefore, emerge as new potential therapeutic targets in the management of psychiatric disorders, stroke, neurodegenerative diseases and pain.     

Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells

Recombinant human P2X(7) receptors, C-terminally labelled with enhanced green fluorescent protein (P2X(7)-EGFP), were transiently expressed in HEK293 cells. Activation of these receptors by their preferential agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) induced inward currents and propidium ion uptake indicating the opening of cationic channels and of large pores permeable for dye molecules, respectively. Two mutants of P2X(7) receptors (P2X(7)-EGFP-I568N, -E496A) representing polymorphisms in the P2X(7) gene known to interfere with normal receptor-trafficking and with optimal assembly of its subunits, responded with much lower current amplitudes to BzATP than their wild-type counterpart. Similarly, the normal propidium ion uptake induced by BzATP at the wild-type P2X(7) receptor was abolished by the two mutants. Confocal laser scanning microscopy indicated that in vitro ischemia of 12h duration increased the integration of P2X(7)-EGFP, but not of its two mutants, into the plasma membrane of HEK293 cells. Further, this ischemic stimulus facilitated the current response to BzATP in HEK293 cells permanently transfected with P2X(7) receptors. Finally, the fluorescence intensity per cell measured by flow cytometry and P2X(7) antibodies directed against an extracellular, but not an intracellular epitope of the receptor, were also increased. In conclusion, P2X(7) receptors may alter their trafficking properties during ischemia and thereby contribute to the ATP-induced damage of various cell-types including neurons.     

Insights into the channel gating of P2X receptors from structures,dynamics and small molecules

P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na⁺, K⁺ and Ca²⁺. Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors.     

The signalling role of action potential depolarization in insulin secretion: Metabolism-dependent dissociation between action potential increase and secretion increase by TEA

The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10 mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5 mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1 μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.     

ATP-gated P2X cation-channels

P2X receptors are ATP-gated cation channels with important roles in diverse pathophysiological processes. Substantial progress has been made in the last few years with the discovery of both subunit selective antagonists and modulators. The purpose of this brief review is to summarize the advances in the pharmacology of P2X receptors, with key properties presented in an easy to access format. Ligand-gated ion channels consist of three families in mammals; the ionotropic glutamate receptors, the Cys-loop receptors (for GABA, ACh, glycine and serotonin) and the P2X receptors for ATP. The first two of these are considered in articles accompanying this Special Issue. Here we consider the pharmacological properties of P2X receptors. We do not present a detailed discussion of P2X receptor physiological roles or structure-function studies. Moreover, the pharmacological basis for discriminating between the main subtypes of P2X receptor and their nomenclature has been published by the Nomenclature Committee of the International Union of Pharmacology (NC-IUPHAR) P2X Receptor Subcommittee, and so these aspects are not revisited here. Instead in this brief article we seek to present a summary of the pharmacology of recombinant homomeric and heteromeric P2X receptors, with particular emphasis on new antagonists. In this article we have tried to present as much information as possible in two tables in the hope this will be useful as a day-to-day resource, and also because an excellent and detailed review has recently been published.     

Potential therapeutic targets for ATP-gated P2X receptor ion channels

P2X receptors make up a novel family of ligand-gated ion channels that are activated by binding of extracellular ATP. These receptors can form a number of homomeric and heteromeric ion channels, which are widely distributed throughout the human body. They are thought to play an important role in many cellular processes, including synaptic transmission and thrombocyte aggregation. These ion channels are also involved in the pathology of several disease states, including chronic inflammation and neuropathic pain, and thus are the potential targets for drug development. The recent discovery of potent and highly selective antagonists for P2X(7) receptors, through the use of high-throughput screening, has helped to further understand the P2X receptor pharmacology and provided new evidence that P2X(7) receptors play a specific role in chronic pain states. In this review, we discuss how the P2X family of ion channels has distinguished itself as a potential new drug target. We are optimistic that safe and effective candidate drugs will be suitable for progression into clinical development.     

Go it alone no more--P2X7 joins the society of heteromeric ATP-gated receptor channels

P2X receptors (P2XR) function as ATP-gated nonselective ion channels permeable to Na+, K+, and Ca2+, and they are expressed in a wide range of excitable, epithelial/endothelial, and immune effector cell types. The channels are trimeric complexes composed of protein subunits encoded by seven different P2XR genes expressed in mammalian and other vertebrate genomes. Current genetic, biochemical, and/or physiological evidence indicates that the extended family of functional P2X receptors includes six homomeric channels composed of P2X1, P2X2, P2X3, P2X4, P2X5, or P2X7 subunits and six heteromeric channels that involve subunit pairings of P2X1/P2X2, P2X1/P2X4, P2X1/P2X5, P2X2/P2X3, P2X2/P2X6, or P2X4/P2X6. Thus, all P2XR subtypes--with the salient exception of P2X7R--have previously been implicated in the assembly of heteromeric ATP-gated ion channels that can comprise unique pharmacological targets in different tissues. The assumed "go-it alone" function of the P2X7R has important implications because agents that target this particular receptor have been proposed as useful therapeutics in various autoinflammatory diseases or amelioration of inflammatory pain. However, this assumption and the interpretations based on it now require reevaluation in light of a new report in this issue of Molecular Pharmacology (p. 1447) that provides convincing biochemical and electrophysiological evidence for the existence of P2X4/P2X7 heteromeric receptors.     

芋螺镇痛多肽研究进展

芋螺多肽由芋螺毒液管和毒囊内壁的毒腺所分泌,大多数芋螺多肽由10～40个氨基酸残基组成,且富含二硫键,能特异性作用于乙酰胆碱受体 (nAChR),及钙、钠、钾等多种离子通道亚型。目前已发现作用于N-型钙通道、nAChR的α9α10亚基、TTX-R钠通道、NMDA受体的芋螺多肽具有很强的镇痛活性,其中N-型钙通道抑制剂ω-MVIIA已于2004年上市。该类镇痛多肽具有相对分子质量小、结构稳定、活性及选择性高等特点。芋螺镇痛多肽不仅会成为镇痛机制等相关神经生物学研究的重要工具,也会为开发新一代无致瘾镇痛药起到重要作用。本文对芋螺镇痛多肽研究的最新进展予以评述,着重介绍芋螺镇痛多肽的作用靶位、构效关系及其应用进展。     

Involvement of uracil nucleotides in protection of cardiomyocytes from hypoxic stress

Cardiomyocytes express one or more subtypes of P2 receptors for extracellular nucleotides. P2 purinoceptors, which are activated by nucleotides, are classified as P2X or P2Y: P2X receptors are ligand-gated intrinsic ion channels, and P2Y receptors are G protein-coupled receptors. Extracellular pyrimidine and purine nucleotides are released from the heart during hypoxia. Although the cardioprotective effects of purines acting via purinoceptors were studied intensively, the physiological role of uracil nucleotide-responsive P2Y2, P2Y4, P2Y6, and P2Y14 receptors is still unclear, especially in the cardiovascular system. This study revealed that uridine-5'-triphosphate (UTP) protected cultured rat cardiomyocytes during hypoxia and explored the UTP signaling pathway leading to this cardioprotection. We found that UTP, but not UDP or uridine, significantly reduced cardiomyocyte death induced by hypoxia. Incubation with UTP for 1 h, before exposure to hypoxic conditions, protected the cells 24 h later. The cardioprotective effect of UTP was reduced in the presence of the P2 antagonist suramin. In addition, UTP caused a transient increase of [Ca2+]i in cardiomyocytes. Pyridoxal-5'-phosphate-6-azophenyl-2,4-disulfonate (PPADS) or Reactive blue 2 (RB-2), other antagonists of P2 receptors, abolished the [Ca2+]i elevation caused by UTP. We used various inhibitors of the Ca2+ signaling pathway to show that UTP elevated levels of [Ca2+]i, originating from intracellular sources, via activation of phospholipase C and the IP3 receptor. Interestingly, these inhibitors of the Ca2+ signaling pathway did not prevent the immediate protective effect caused by UTP. Although mitochondrial KATP channels are involved in other preconditioning mediator pathways, the involvement of these channels in the cardioprotective effect induced by UTP was ruled out, because 5-hydroxydecanoic acid (5-HD), a specific inhibitor of these channels, did not prevent the protection.