The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.
The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.
Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular loop. Functionally, P2X5 and P2X6 receptors most resemble P2X2 and P2X4; they desensitize only slowly and do not respond to alpha beta methylene-ATP. P2X6 receptors, like P2X4, receptors, are not blocked by the antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. P2X6 and P2X5 receptors express at lower levels than P2X1-P2X4 receptors do, perhaps indicating that they do not normally form homomultimeric channels. P2X6 and P2X4 are the receptors expressed most heavily in brain, where their RNAs have a widespread and extensively overlapping distribution. The spinal cord expresses all receptors except P2X3. P2X2, P2X4, and P2X6, are the most abundant in the dorsal horn. Sensory neurons of the trigeminal, dorsal root, and nodose ganglia express all six RNAs; P2X3 is found only there. The functional properties and tissue distribution of these six P2X receptors indicate new roles for ATP-gated ion channels.
Cation-selective P2X receptor channels were first described in sensory neurons where they are important for primary afferent neurotransmission and nociception. Here we report the cloning of a complementary DNA (P2X3) from rat dorsal root ganglia that had properties dissimilar to those of sensory neurons. We also found RNA for (P2X1)(ref. 7), (P2X2)(ref. 8) and P2X4 (ref. 9) in sensory neurons; channels expressed from individual cDNAs did not reproduce those of sensory ganglia. Coexpression of P2X3 with P2X2, but not other combinations, yielded ATP-activated currents that closely resembled those in sensory neurons. These properties could not be accounted for by addition of the two sets of channels, indicating that a new channel had formed by subunit heteropolymerization. Although in some tissues responses to ATP can be accounted for by homomeric channels, our results indicate that ATP-gated channels of sensory neurons may form by a specific heteropolymerization of P2X receptor subunits.
Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels.
The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X(7) receptor, via regulation of mature IL-1beta production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad-spectrum analgesia.
ATP is a known mediator of inflammatory and neuropathic pain. However, the mechanisms by which specific purinergic receptors contribute to chronic pain states are still poorly characterized. Here, we demonstrate that in response to peripheral nerve injury, P2X 4 receptors (P2X 4 R) are expressed de novo by activated microglia in the spinal cord. Using in vitro and in vivo models, we provide direct evidence that P2X 4 R stimulation leads to the release of BDNF from activated microglia and, most likely phosphorylation of the NR1 subunit of NMDA receptors in dorsal horn neurons of the spinal cord. Consistent with these findings, P2X4-deficient mice lack mechanical hyperalgesia induced by peripheral nerve injury and display impaired BDNF signaling in the spinal cord. Furthermore, ATP stimulation is unable to stimulate BDNF release from P2X 4 -deficient mice microglia in primary cultures. These results indicate that P2X 4 R contribute to chronic pain through a central inflammatory pathway. P2X 4 R might thus represent a potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain injury and neurodegenerative disorders.
A cDNA was cloned which encodes a new ATP‐gated ion channel (P2X4 receptor). ATP induces a cationic current in HEK293 cells transfected with the P2X4 receptor. However, the current is almost completely insensitive to antagonists effective at other P2X receptors. Sensitivity to two of these antagonists (pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid and pyridoxal 5‐phosphate) is restored by replacement of Glu249 by lysine, which occurs at the equivalent position in P2X1 and P2X2 receptors. P2X4 RNA is found by in situ hybridization in the brain, peripheral ganglia and epithelia including serosal cells of salivary glands. Recordings from rat submandibular gland cells showed ATP‐induced currents that are also insensitive to antagonists. These results define a further member of P2X receptor family, and they identify an amino acid residue involved in antagonist binding. They also introduce a new phenotype for ATP responses at P2X receptors–insensitivity to currently known antagonists.
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