P2X receptors for adenosine tri-phosphate (ATP) are a distinct family of ligand-gated cation channels with two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand binding loop. Seven genes (P2X(1-7)) have been cloned and the channels form as either homo or heterotrimeric channels giving rise to a wide range of phenotypes. This review aims to give an account of recent work on the molecular basis of the properties of P2X receptors. In particular, to consider emerging information on the assembly of P2X receptor subunits, channel regulation and desensitisation, targeting, the molecular basis of drug action and the functional contribution of P2X receptors to physiological processes.

No abstract

P2X receptors for extracellular ATP are a distinct family of ligand-gated cation channels involved in physiological processes ranging from synaptic transmission to muscle contraction. Common ATP binding motifs are absent from P2X receptors, and the extent of the agonist binding site is unclear. We used cysteine-scanning mutagenesis, radiolabeled 2-azido ATP binding, and methanethiosulfonate (MTS) compounds to identify amino acid residues involved in ATP binding and gating of the human P2X 1 receptor. The pattern of MTSEA [(2-aminoethyl)methanethiosulfonate hydrobromide] biotinylation was also used to determine the accessibility of substituted cysteine residues and whether this changed on addition of ATP. Analysis of cysteine-substituted mutants of the last 44 amino acid residues (S286 -I329) in the extracellular loop before the second transmembrane segment showed that N290, F291, R292, and K309 mutants had reduced ATP potency and 2-azido ATP binding. MTS reagents produced additional shifts in ATP potency at these residues, suggesting that they are directly involved in ATP binding; the effects were dependent on the charge of the MTS reagent at K309C; one explanation for this is that K309 interacts directly with the negatively charged phosphate of ATP. The remainder of the cysteine substitutions had little or no effect on ATP potency. However, at the mutants D316C, G321C, A323C, and I328C, MTS reagents did not change ATP potency but modified agonist-evoked responses, suggesting that this region may contribute to the gating of the channel.

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X 1 receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X 2 and P2X 4 receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X 2 K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action.P2X receptors for ATP comprise a distinct family of ligandgated ion channels, with two transmembrane domains, intracellular amino and carboxyl termini and a large extracellular ligand binding loop. Seven P2X receptor genes have been identified (P2X 1-7 ) and the subunits form functional homo-and heterotrimeric ion channels with a variety of phenotypes (1). ATP is released from neurons, in response to shear stress, as well as from damaged cells. P2X receptors have been shown to be involved in a wide range of physiological roles including blood clotting (2), sensory perception (including pain (3-5), bladder voiding (6), and taste (7)), as well as bone formation (8).As the receptors provide novel drug targets for a range of diseases (9) an understanding of the agonist binding site would be useful for rational drug design. However, it is clear that common ATP binding sequences, e.g. the Walker motif (10), are not present in P2X receptors. In the absence of a crystal structure a site-directed mutagenesis approach has been used to gain insight into amino acids important in mediating the actions of ATP at P2X receptors.We have used the human P2X 1 receptor and alanine mutagenesis of conserved residues in the extracellular loop ( Fig. 1) to propose a model of the ATP binding site (reviewed in Refs. 11 and 12). Cysteine substitution mutagenesis and charged methanethiosulfonate (MTS) 3 compounds have been useful in investigating a variety of ion channels including the pore region (13, 14) as well as...

1 The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca 2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2 Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca 2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca 2+ had little eect on the peak Ca 2+-response, but resulted in a more rapid decline to basal. There was no response to a,b-MethylATP (a,bMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3 ATP (log EC 50 75.1+0.2) also caused an increase in the total [3 H]-inositol (poly)phosphates ([ 3 H]-InsP x ) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP4ADP, with 2MeSATP and a,bMeATP giving no detectable response. 4 Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P 3 .5 None of the nucleotides tested aected basal cyclic AMP, while ATP and ATPgS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 mM forskolin. 6 Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentrationdependency consistent with activation at P2Y 2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP. 7 These results are consistent with brain endothelial regulation by P2Y 2 receptors coupled to phospholipase C, Ca 2+ and MAPK; and by P2Y 1 -like (2MeSATP-sensitive) receptors which are linked to Ca 2+ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P 3 levels. A further response to ATP, acting at an unde®ned receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The dierential MAPK coupling of these receptors suggests that they exert fundamentally distinct in¯uences over brain endothelial function.