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.

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.

P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by approximately 90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally--reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility.

1. Complementary DNAs for the ATP-gated ion channel subunits P2X, (from human bladder) and P2X2 (from rat phaeochromocytoma (PC12) cells) were used to express the receptors in human embryonic kidney cells by stable transfection, and in Chinese hamster ovary cells by viral infection. 2. Membrane currents evoked by ATP were recorded by the whole-cell patch clamp method.The reversal potential of the current was measured with various intracellular and extracellular solutions and used to compute the relative permeability of the P2X receptor channels. 3. There was no difference between the two receptors with respect to their permeability to monovalent organic cations. The relative permeabilities (Px/PNa) were 2-3, 1P0, 1 0, 0 95, 0'72, 0 5, 0-29, 0-16, 0 04 and 0 03 for guanidinium, potassium, sodium, methylamine, caesium, dimethylamine, 2-methylethanolamine, tris(hydroxymethyl)-aminomethane, tetraethylammonium and N-methyl-D-glucamine, respectively (values for P2X2 receptor). 4. The calcium permeability of P2X1 receptors was greater than that of P2X2 receptors. Under biionic conditions (112 mm calcium outside, 154 mm sodium inside), Pca/PNa values were 3 9 and 2 2, respectively (corrected for ionic activities). 5. ATP-evoked currents in cells expressing the P2X2 receptor were strongly inhibited when the extracellular calcium concentration was increased (0 3-30 mM); the action of ATP could be restored by increasing the ATP concentration. ATP-evoked currents in cells expressing the P2X1 receptor were not inhibited by such increases in the extracellular calcium concentration.

Summary.A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudornonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P . cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P . cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could compete with transferrin for iron. Azurechelin could also stimulate iron uptake by P . cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.

1 P2X receptor activation by a,b-meATP evoked inward currents in acutely dissociated rat mesenteric artery smooth muscle cells and contractions of whole artery rings. 2 The selective P2X 1 and P2X 3 receptor antagonist TNP-ATP inhibited P2X receptor mediated inward currents in response to 3 mM a,b-meATP (an *EC 90 concentration) with an IC 50 of *2 nM. This provides further evidence that the P2X receptor underlying membrane depolarisation associated with P2X receptor activation can be accounted for by the expression of P2X 1 receptors. 3 TNP-ATP inhibited a,b-meATP induced contractions with an IC 50 of *30 mM and had non-speci®c e ects on smooth muscle contraction. 4 The reduced potency of TNP-ATP in whole tissue experiments probably re¯ects the breakdown of TNP-ATP by nucleotidases. Thus, TNP-ATP is of limited use in whole tissue experiments as a P2X receptor antagonist.

1 Immunoreactivity for P2X 1 , P2X 4 and P2X 5 receptor subtypes was detected in the smooth muscle cell layer of second and third order rat mesenteric arteries immunoreactivity, for P2X 2 , P2X 3 , P2X 6 and P2X 7 receptors was below the level of detection in the smooth muscle layer. 2 P2X receptor-mediated currents were recorded in patch clamp studies on acutely dissociated mesenteric artery smooth muscle cells. Purinergic agonists evoked transient inward currents that decayed rapidly in the continued presence of agonist (t*200 ms). Standard whole cell responses to repeated applications of agonist at 5 min intervals ran down. Run-down was unaected by changes in extracellular calcium concentration, intracellular calcium buering or the inclusion of ATP and GTP in the pipette solution. 3 Run-down was overcome and reproducible responses to purinergic agonists were recorded using the amphotericin permeabilized patch recording con®guration. 4 The rank order of potency at the P2X receptor was ATP=2 methylthio ATP4a,b-methylene ATP4CTP=l-b,g-methylene ATP. Only ATP and 2meSATP were full agonists. The P2 receptor antagonists suramin and PPADS inhibited P2X receptor-mediated currents with IC 50 s of 4 mM and 70 nM respectively. 5 These results provide further characterization of artery P2X receptors and demonstrate that the properties are dominated by a P2X 1 -like receptor phenotype. No evidence could be found for a phenotype corresponding to homomeric P2X 4 or P2X 5 receptors or to heteromeric P2X

The expression of the seven P2X receptor subunits (P2X_1–7) in the rat vascular system was determined using subtype-selective antibodies. Arteries of different sizes (from arterioles to conduit vessels) from a range of vascular beds were used to give an overview of receptor expression. P2X₁ receptor immunoreactivity was detected in the smooth muscle layer of arteries. The relative level of P2X₁ receptor immunoreactivity was dependent on the size of the artery and the vascular bed; expression was highest in small and medium arteries. P2X₄ receptors were detected in all arteries; once again, the relative level of expression was dependent on the size of the artery and the vascular bed. P2X₅ receptor immunoreactivity was barely detectable in most arteries studied. P2X₇ receptor immunoreactivity was generally punctate and associated with the outer adventitial layer. Immunoreactivity for P2X₂, P2X₃ and P2X₆ receptors was not detected in arteries. These results demonstrate that arteries express multiple P2X receptor subunits and that there is a heterogeneity in the level of expression. The properties of artery P2X receptors correspond to homomeric P2X₁ receptors, and the function of P2X₄ and P2X₅ receptor subunits in arteries is unclear.