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Title: Use of chimeras, point mutants, and molecular modeling to map the antagonist-binding site of 4,4',4″,4‴-(carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF449) at P2X1 receptors for ATP.
Authors: Farmer, Louise K.
Schmid, Ralf
Evans, Richard J.
First Published: 16-Jan-2015
Publisher: American Society for Biochemistry and Molecular Biology
Citation: Journal of Biological Chemistry, 2015, 290 (3), pp. 1559-1569
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.
DOI Link: 10.1074/jbc.M114.592246
ISSN: 0021-9258
eISSN: 1083-351X
Version: Post-print
Status: Peer-reviewed
Type: Journal Article
Rights: Archived with reference to SHERPA/RoMEO and publisher website. This research was originally published in Journal of Biological Chemistry. Louise K. Farmer, Ralf Schmid and Richard J. Evans. Use of Chimeras, Point Mutants, and Molecular Modeling to Map the Antagonist-binding Site of 4,4′,4″,4‴-(Carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic Acid (NF449) at P2X1 Receptors for ATP. Journal of Biological Chemistry. 2015. 290 (3), pp. 1559-1569. © the American Society for Biochemistry and Molecular Biology
Description: PMCID: PMC4340402
Appears in Collections:Published Articles, Dept. of Cell Physiology and Pharmacology

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