The P2X7 receptor (P2X7R) for ATP is a therapeutic target for pathophysiological states including inflammation, pain management and epilepsy. This is facilitated by the predicted low side effect profile as the high concentrations of ATP required to activate the receptor are usually only found following cell damage/disease and so P2X7Rs respond to a “danger” signal and are not normally active. AZ10606120 is a selective antagonist for P2X7Rs (IC50 of ~10 nM) and ineffective at the P2X1R (at 10 μM). To determine the molecular basis of selectivity we generated a series of P2X7/1R chimeras and mutants. Two regions that are unique to the P2X7R, a loop insertion (residues 73–79) and threonine residues T90 and T94, are required for high affinity antagonist action. Point mutations ruled out an orthosteric antagonist site. Mutations and molecular modelling identified an allosteric binding site that forms at the subunit interface at the apex of the receptor. Molecular dynamics simulations indicated that unique P2X7R features regulate access of AZ10606120 to the allosteric site. The characterisation of the allosteric pocket provides a new and novel target for rational P2X7R drug development.

P2X receptors for ATP have a wide range of physiological roles and comprise a structurally distinct family of ligand-gated trimeric ion channels. The crystal structure of a P2X4 receptor, in combination with mutagenesis studies, has provided a model of the intersubunit ATP-binding sites and identified an extracellular lateral portal, adjacent to the membrane, that funnels ions to the channel pore. However, little is known about the extent of ATP-induced conformational changes in the extracellular domain of the receptor. To address this issue, we have used MTSEA-biotinylation (N-Biotinoylaminoethyl methanethiosulfonate) to show ATP-sensitive accessibility of cysteine mutants at the human P2X1 receptor. Mapping these data to a P2X1 receptor homology model identifies significant conformational rearrangement. Electron microscopy of purified P2X1 receptors showed marked changes in structure on ATP binding, and introducing disulphide bonds between adjacent subunits to restrict intersubunit movements inhibited channel function. These results are consistent with agonist-induced rotation of the propeller-head domain of the receptor, sliding of adjacent subunits leading to restricted access to the upper vestibule, movement in the ion conducting lateral portals, and gating of the channel pore.purinergic receptor | cysteine-scanning A TP functions not only as an energy carrier within cells but also acts extracellularly at P2 receptors (G protein-coupled P2Y receptors and ligand-gated P2X receptor cation channels) (1) to mediate signaling between cells. A role for purinergic signaling was first suggested in the 1970s (2) and it is now known that one or more P2X receptor subtypes are expressed on virtually every cell type in the body (3, 4). Given the almost ubiquitous expression of the receptors, it is not surprising that they have been shown to have widespread physiological roles, ranging from taste sensation to bone formation (3, 5). In addition, P2X receptors contribute to pathophysiological states, including thromboembolism (6) and pain (7), highlighting the considerable therapeutic potential of P2X receptor-selective drugs (4).Genes encoding seven mammalian P2X receptors have been identified (P2X1 to -7). Functional receptors form from the homo-or heterotrimeric assembly of subunits with intracellular amino and carboxyl termini, two transmembrane segments, and a large extracellular ligand-binding loop (8). This topology and stoichiometry is different from that of the cys loop and glutamate gated ion channels (9). The crystalisation of a zebrafish P2X4 receptor in an ATP-free, closed, resting state (10) provided a major advance in understanding the molecular basis of receptor function. The individual subunits have been likened to the shape of a dolphin (Fig. S1), with the transmembrane domains corresponding to the fluke and the extracellular region to the body, with a cysteine-rich head domain, flippers, and a dorsal fin. The three subunits are entwined in a right-handed twist surrounding the upper, central, and extracel...

Background: P2X receptors show marked variation in the time-course of responses.Results: Amino-terminal chimeric and mutant P2X1 and -2 receptors had reciprocal effects on desensitization.Conclusion: The intracellular amino terminus plays a dominant role in regulation of the time-course of ATP currents.Significance: The P2X receptor channel pore region formed by the transmembrane segments is regulated by the amino terminus.

P2X receptors are ATP-gated cation channels. The x-ray structure of a P2X4 receptor provided a major advance in understanding the molecular basis of receptor properties. However, how agonists are coordinated, the extent of the binding site, and the contribution of the vestibules in the extracellular domain to ionic permeation have not been addressed. We have used cysteine-scanning mutagenesis to determine the contribution of residues Glu52–Gly96 to human P2X1 receptor properties. ATP potency was reduced for the mutants K68C, K70C, and F92C. The efficacy of the partial agonist BzATP was also reduced for several mutants forming the back of the proposed agonist binding site. Molecular docking in silico of both ATP and BzATP provided models of the agonist binding site consistent with these data. Individual cysteine mutants had no effect or slightly increased antagonism by suramin or pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate. Mutants at the entrance to and lining the upper vestibule were unaffected by cysteine-reactive methanethiosulfonate (MTS) reagents, suggesting that it does not contribute to ionic permeation. Mutants that were sensitive to modification by MTS reagents were predominantly found either around the proposed ATP binding pocket or on the strands connecting the binding pocket to the transmembrane region and lining the central vestibule. In particular, ATP sensitivity and currents were increased by a positively charged MTS reagent at the G60C mutant at the interface between the central and extracellular vestibule. This suggests that dilation of the base of the central vestibule contributes to gating of the receptor.

Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20–23 and 27–29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.