Abstract
P2X receptor subtype-selective antagonists are promising candidates for treatment of a range of pathophysiological conditions. However, in contrast to high resolution structural understanding of agonist action in the receptors, comparatively little is known about the molecular basis of antagonist binding. We have generated chimeras and point mutations in the extracellular ligand-binding loop of the human P2X1 receptor, which is inhibited by NF449, suramin, and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate, with residues from the rat P2X4 receptor, which is insensitive to these antagonists. There was little or no effect on sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate in chimeric P2X1/4 receptors, indicating that a significant number of residues required for binding of these antagonists are present in the P2X4 receptor. Sensitivity to the P2X1 receptor-selective antagonist NF449 was reduced by ∼60- and ∼135-fold in chimeras replacing the cysteine-rich head, and the dorsal fin region below it in the adjacent subunit, respectively. Point mutants identified the importance of four positively charged residues at the base of the cysteine-rich head and two variant residues in the dorsal fin for high affinity NF449 binding. These six residues were used as the starting area for molecular docking. The four best potential NF449-binding poses were then discriminated by correspondence with the mutagenesis data and an additional mutant to validate the binding of one lobe of NF449 within the core conserved ATP-binding pocket and the other lobes coordinated by positive charge on the cysteine-rich head region and residues in the adjacent dorsal fin.
Highlights
NF449 is a selective P2X1 receptor antagonist
Characterization of ATP Action at P2X1/4 Receptor Chimeras—Four chimeras replacing sections of the extracellular loop of the human P2X1 (hP2X1) receptor with the corresponding part of the rat P2X4 (rP2X4) receptor were designed with the aid of sequence lineups and homology models
The chimeras between the hP2X1 and rP2X4 receptors in this study have been useful to identify whether a region contributes to differences in properties, e.g. ATP sensitivity and desensitization
Summary
NF449 is a selective P2X1 receptor antagonist. Results: Chimeric/mutant P2X1/4 receptors and molecular docking provided models of NF449 binding. Sensitivity to the P2X1 receptor-selective antagonist NF449 was reduced by ϳ60- and ϳ135-fold in chimeras replacing the cysteine-rich head, and the dorsal fin region below it in the adjacent subunit, respectively. Point mutants identified the importance of four positively charged residues at the base of the cysteine-rich head and two variant residues in the dorsal fin for high affinity NF449 binding These six residues were used as the starting area for molecular docking. The four best potential NF449-binding poses were discriminated by correspondence with the mutagenesis data and an additional mutant to validate the binding of one lobe of NF449 within the core conserved ATP-binding pocket and the other lobes coordinated by positive charge on the cysteine-rich head region and residues in the adjacent dorsal fin. Site-directed mutagenesis was used to identify individual residues that contribute to NF449 action, which were the starting point for in silico antagonist docking and validation
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