title: Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel.
abstract: P2X receptors for ATP have a wide range of physiological roles and comprise a structurally distinct family of ligand-gated trimeric ion channels. The crystal structure of a P2X4 receptor, in combination with mutagenesis studies, has provided a model of the intersubunit ATP-binding sites and identified an extracellular lateral portal, adjacent to the membrane, that funnels ions to the channel pore. However, little is known about the extent of ATP-induced conformational changes in the extracellular domain of the receptor. To address this issue, we have used MTSEA-biotinylation (N-Biotinoylaminoethyl methanethiosulfonate) to show ATP-sensitive accessibility of cysteine mutants at the human P2X1 receptor. Mapping these data to a P2X1 receptor homology model identifies significant conformational rearrangement. Electron microscopy of purified P2X1 receptors showed marked changes in structure on ATP binding, and introducing disulphide bonds between adjacent subunits to restrict intersubunit movements inhibited channel function. These results are consistent with agonist-induced rotation of the propeller-head domain of the receptor, sliding of adjacent subunits leading to restricted access to the upper vestibule, movement in the ion conducting lateral portals, and gating of the channel pore.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Roberts
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Allsopp
authors.forename: Sam
authors.initials: S
authors.lastname: El Ajouz
authors.forename: Catherine
authors.initials: C
authors.lastname: Vial
authors.forename: Ralf
authors.initials: R
authors.lastname: Schmid
authors.forename: Mark T
authors.initials: MT
authors.lastname: Young
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Evans
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D007477:Ions / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods; D008968:Molecular Conformation; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D058474:Receptors, Purinergic P2X1 / Q000737:chemistry*; D014981:Xenopus
chemical_list: D004220:Disulfides; D007477:Ions; D058474:Receptors, Purinergic P2X1; D000255:Adenosine Triphosphate