title: Amino acid residues constituting the agonist binding site of the human P2X3 receptor.
abstract: Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.
authors.affiliation: Rudolf-Boehm Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Mandy
authors.initials: M
authors.lastname: Bodnar
authors.forename: Haihong
authors.initials: H
authors.lastname: Wang
authors.forename: Thomas
authors.initials: T
authors.lastname: Riedel
authors.forename: Stefan
authors.initials: S
authors.lastname: Hintze
authors.forename: Erzsebet
authors.initials: E
authors.lastname: Kato
authors.forename: Ghada
authors.initials: G
authors.lastname: Fallah
authors.forename: Helke
authors.initials: H
authors.lastname: Gröger-Arndt
authors.forename: Rashid
authors.initials: R
authors.lastname: Giniatullin
authors.forename: Marcus
authors.initials: M
authors.lastname: Grohmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Hausmann
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
authors.forename: Peter
authors.initials: P
authors.lastname: Illes
authors.forename: Patrizia
authors.initials: P
authors.lastname: Rubini
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D009865:Oocytes; D010449:Peptide Mapping; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058911:Purinergic P2X Receptor Agonists / Q000737:chemistry* / Q000378:metabolism; D058477:Receptors, Purinergic P2X3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D021122:Protein Subunits; D058911:Purinergic P2X Receptor Agonists; D058477:Receptors, Purinergic P2X3