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.

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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A cDNA was isolated from a human monocyte library that encodes the P2X 7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2,3-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X 7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X 7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.Cell surface receptors for ATP can be divided into metabotropic (P2Y/P2U) and ionotropic (P2X) classes. The metabotropic class belong to the superfamily of G protein-coupled receptors with seven transmembrane segments; the ionotropic class are ligand-gated channels, currently thought to be multisubunit proteins with two transmembrane domains per subunit (for review see Ref. 1). P2Z receptors have been distinguished from other P2 receptors in three main ways (2-4). First, activation of this receptor leads not only to an inward ionic current but also to cell permeabilization. Second, 2Ј,3Ј-(4-benzoyl)benzoyl ATP is the most effective agonist, and ATP itself is of rather low potency. Third, responses are strongly inhibited by extracellular magnesium ions, which has been interpreted to indicate that ATP Ϫ4 is the active agonist (for review see Ref. 5).A seventh member of the P2X receptor family was isolated recently from a rat cDNA library that, when expressed in human embryonic kidney (HEK293) cells, exhibits these three properties (6). This receptor (rP2X 7 ) 1 is thus considered to represent a P2Z receptor. The protein is structurally related to other members of the P2X family; there is 35-40% amino acid identity in the region of homology, but the C terminus is 239 amino acids long in the rP2X 7 receptor compared with 27-120 amino acids in the others. The rP2X 7 receptor functions both as a channel permeable to small cations and as a cytolytic pore (6). Brief applications of ATP (1-2 s) transiently open the channel, and this is generally similar in properties to other P2X receptors. Repeated or prolonged applications of agonist cause cell permeabilization; reducing the extracellular magnesium concentration much potentiates this effect. The permeabilization involves the cytoplasmic C terminus of the protein because it does not occur with a P2X 7 receptor lacking the last 177 residues, although this truncation does not affect the function as a small cation channel. The P2Z receptor has been implicated in lysis of antigenpresenting cells by cytotoxic T lymphocytes, in the mi...

What are P2X receptors?P2X receptors are ligand-gated ion channels activated by ATP. Early evidence for the role of extracellular ATP in signalling was based on the ability of ATP and its analogues to contract smooth muscle; this work has been reviewed by Burnstock (1980). Subsequently, ATP was shown to gate channels on a subset of sensory neurons (Jahr and Jessell, 1983). These and other observations have led to the identification of distinct P2 purinergic receptors which are divided into metabotropic (P2Y) and ionotropic families (P2X and P2Z) (Abbracchio and Bumstock, 1994;Fredholm et al., 1994). Several P2Y receptors have been cloned (Barnard et al., 1994); they display seven transmembrane structures and signal via a variety of second messenger systems, including inositol triphosphate production and the inhibition of CAMP .P2Z receptors are channel-forming proteins present on the surface of macrophages (Nuttle et al., 1993;Wiley et al., 1994;Bretschneider et al., 1995). Since the activation of P2Z receptors induces the formation of large pores, which results in cell lysis, the physiological role of these receptors has remained unresolved.Cloning of cDNA has recently shown that P2X receptors constitute a new class of channel-forming proteins which are structurally distinct from other ligand-gated channels, such as those for glutamate or acetylcholine (Brake et al., 1994;Valera et al., 1994). Cloned P2X receptors form rapidly activated, non-selective cationic channels which are activated by micromolar concentrations of extracellular ATP.The extent of P2X receptor diversity in structure, distribution and phenotype is being currently addressed. This review looks at answers that have been obtained with the molecularly cloned receptors and at the relationship between P2X and P2Z. What factors complicate the study of P2X receptors?Establishing the physiological role of extracellular ATP in signalling has been slowed by several constraints. First, purinergic agonists specific for P2X receptors have not yet been found. ATP is a relatively labile agonist, rapidly degraded by ectonucleotidases to ADP, and eventually to adenosine; both products may in turn activate their own receptors. Agonist degradation has also hampered the discrimination of P2 receptor subtypes. For example a$-methylene ATP (a,PmeATP), a stable ATP analogue, is more active than ATP in intact smooth muscle preparations (Burnstock and Kennedy, 1985). However, the opposite agonist order (ATP >> ct,pmeATP) is observed for the cloned smooth muscle receptor (P2X1) (Evans et al., 1995) when it is expressed in systems that are essentially devoid of nucleotidase activity.Second, all cells have an abundance of ATP-binding proteins that may interfere with receptor binding analysis of P2X receptors (Humphrey el al., 1995). As a result there is relatively little receptor autoradiographic data on the distribution of P2X receptors. Only work with the relatively selective agonist [3H]a,PmeATP has met with some success in identifying binding sites in the rat brain (B...

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion.

Progressive degeneration and intraneuronal Lewy bodies made of filamentous a-synuclein (a-syn) in dopaminergic cells of the nigrostriatal system are characteristics of Parkinson's disease (PD). Glucose uptake is reduced in some of the brain regions affected by PD neurodegenerative changes. Defects in mitochondrial activity in the substantia nigra have been observed in the brain of patients affected by PD and substantia nigra lesions can induce the onset of a secondary parkinsonism. Thus, energy starvation and consequently metabolic impairment to dopaminergic neurons may be related to the onset of PD. On this line, we evaluated the effect of nutrient starvation, reproduced 'in vitro' by glucose deprivation (GD), in primary mesecephalic neuronal cultures and dopaminergic-differentiated SH-SY5Y cells, to evaluate if decreased glucose support to dopaminergic cells can lead to mitochondrial damage, neurodegeneration and a-syn misfolding. Furthermore, we investigated the effect of dopamine (DA) treatment in the presence of a DA-uptake inhibitor or of the D 2 /D 3 receptor (D 2 R/D 3 R) agonist quinpirole on GDtreated cells, to evaluate the efficacy of these therapeutic compounds. We found that GD induced the formation of fibrillary aggregated a-syn inclusions containing the DA transporter in dopaminergic cells. These alterations were accompanied by dopaminergic cell death and were exacerbated by DA overload. Conversely, the block of DA uptake and D 2 R/D 3 R agonist treatment exerted neuroprotective effects. These data indicate that glucose starvation is likely involved in the induction of PD-related pathological changes in dopaminergic neurons. These changes may be counteracted by the block of DA uptake and by dopaminergic agonist treatment.