Use of chimeras, point mutants and molecular modelling to map the antagonist binding site of 4,4′,4”,4”’-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) at P2X1 receptors for ATP [Molecular Biophysics]
November 25th, 2014 by Farmer, L. K., Schmid, R., Evans, R. J.
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 at 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, that is inhibited by NF449, suramin and PPADS, with residues from the rat P2X4 receptor that is insensitive to these antagonists. There was little or no effect on the sensitivity to suramin and PPADS at chimeric P2X1/4 receptors indicating that a significant number of residues required for binding of these antagonists are present at the P2X4 receptor. Sensitivity to the selective P2X1 receptor antagonist NF449 was reduced ~60 and ~135 fold in chimeras replacing the cysteine rich head and the dorsal fin region below it in the adjacent subunit. 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 to 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 co-ordinated by positive charge on the cysteine rich head region and residues in the adjacent dorsal fin.