P2X receptors (P2XR) are trimeric ATP-gated cation channels involved in the fast signal transduction in many cells. Significant advances in the molecular understanding of the P2XR function that have been achieved by the availability of high-resolution structures of the zfP2X4R and, recently, hP2X3R and pdP2X7R [1-4]. However, little is known regarding the molecular determinants of the subtype specific potency and efficacy of orthostericP2XR ligands. We used docking analyses performed athomology models of the rP2X2R and rP2X7R [5,6] to analyze the ligand-binding of ATP and the potent P2X7R agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) that is a weak and partial agonist at the P2X2R. The difference between ATP and BzATP is the 4-benzoylbenzoyl (Bz) moiety present at the ribose of the nucleotide in BzATP. We identified two residues K212 and S284 of the P2X2R that spatially correspond to the P2X7R-specific aromatic residues F218 and F288of the dorsal fin and left flipper domains, respectively. Residues at these positions may coordinate the ribose and Bz moiety of ATP and BzATP, respectively, and determine the different potencies of these ligands at the P2X2R. The K212 and S284 of the P2X2R were substituted by phenylalanine residues to yield rP2X2 RK212F and rP2X2RS284F and were analyzed by TEVC electrophysiology following expression in X. laevis oocytes. Mutating S284 to F in rP2X2Rdid not affect the potency for ATP or BzATP. By contrast, the introduction of an aromatic moiety in position 212 as realized in the P2X2RK212F mutant significantly reduced and strongly increased the potency for ATP and BzATP, respectively. BzATP exhibited a higher potency and efficacy than ATP at the rP2X2RK212F. Our results clearly indicate that an aromatic moiety facing the water-exposed surface in the α3-helix of the P2X2R determines BzATP potency and efficacy, but weakensthe potency and efficacy of ATP.

Identification of a specific residue that confers BzATP sensitivity to P2X2 receptors

DAL BEN, Diego;
2017

Abstract

P2X receptors (P2XR) are trimeric ATP-gated cation channels involved in the fast signal transduction in many cells. Significant advances in the molecular understanding of the P2XR function that have been achieved by the availability of high-resolution structures of the zfP2X4R and, recently, hP2X3R and pdP2X7R [1-4]. However, little is known regarding the molecular determinants of the subtype specific potency and efficacy of orthostericP2XR ligands. We used docking analyses performed athomology models of the rP2X2R and rP2X7R [5,6] to analyze the ligand-binding of ATP and the potent P2X7R agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) that is a weak and partial agonist at the P2X2R. The difference between ATP and BzATP is the 4-benzoylbenzoyl (Bz) moiety present at the ribose of the nucleotide in BzATP. We identified two residues K212 and S284 of the P2X2R that spatially correspond to the P2X7R-specific aromatic residues F218 and F288of the dorsal fin and left flipper domains, respectively. Residues at these positions may coordinate the ribose and Bz moiety of ATP and BzATP, respectively, and determine the different potencies of these ligands at the P2X2R. The K212 and S284 of the P2X2R were substituted by phenylalanine residues to yield rP2X2 RK212F and rP2X2RS284F and were analyzed by TEVC electrophysiology following expression in X. laevis oocytes. Mutating S284 to F in rP2X2Rdid not affect the potency for ATP or BzATP. By contrast, the introduction of an aromatic moiety in position 212 as realized in the P2X2RK212F mutant significantly reduced and strongly increased the potency for ATP and BzATP, respectively. BzATP exhibited a higher potency and efficacy than ATP at the rP2X2RK212F. Our results clearly indicate that an aromatic moiety facing the water-exposed surface in the α3-helix of the P2X2R determines BzATP potency and efficacy, but weakensthe potency and efficacy of ATP.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11581/405726
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