title: Agonist trapped in ATP-binding sites of the P2X2 receptor.
abstract: ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATP-derived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.
authors.affiliation: Laboratoire de Biophysicochimie des Récepteurs Canaux, Unité Mixte de Recherche 7199, Centre National de la Recherche Scientifique, Conception et Application de Molécules Bioactives, Faculté de Pharmacie, Université de Strasbourg, 67400 Illkirch, France.
authors.forename: Ruotian
authors.initials: R
authors.lastname: Jiang
authors.forename: Damien
authors.initials: D
authors.lastname: Lemoine
authors.forename: Adeline
authors.initials: A
authors.lastname: Martz
authors.forename: Antoine
authors.initials: A
authors.lastname: Taly
authors.forename: Sophie
authors.initials: S
authors.lastname: Gonin
authors.forename: Lia
authors.initials: L
authors.lastname: Prado de Carvalho
authors.forename: Alexandre
authors.initials: A
authors.lastname: Specht
authors.forename: Thomas
authors.initials: T
authors.lastname: Grutter
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011487:Protein Conformation; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D058476:Receptors, Purinergic P2X2; D000255:Adenosine Triphosphate; D003545:Cysteine