Amino Acid Residues Constituting the Agonist Binding Site of the Human P2X3 Receptor

Mandy Bodnar,Haihong Wang,Thomas Riedel,Stefan Hintze,Erzsebet Kato,Ghada Fallah,Helke Gröger-Arndt,Rashid Giniatullin,Marcus Grohmann,Ralf Hausmann,Günther Schmalzing,Peter Illes,Patrizia Rubini

doi:10.1074/jbc.m110.167437

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.

Highlights

ATP-gated, cation-permeable P2X receptor channels form a family of seven subunits, referred to as P2X1 through P2X7 [1]
Because in P2X receptors, instead of a few amino acid residues, four clusters of amino acids (AAs), termed nucleotide binding domains (NBD1– 4 [19]; here nucleotide binding segments; NBS1– 4), were identified as possible docking places for ATP, we investigated by Ala scanning mutagenesis the functional significance of these NBSs
Patch Clamp Investigations—The underlined, conserved AA residues in NBS1– 4 of human P2X3 receptor (hP2X3) were sequentially replaced by Ala, one or two at a time (Fig. 1A)

Summary

Introduction

ATP-gated, cation-permeable P2X receptor channels form a family of seven subunits, referred to as P2X1 through P2X7 [1]. Asp or the positively charged Arg. we transfected both the WT receptor and its NBS mutants into HEK293 cells and compared the current responses measured by the patch clamp technique.

Results

Conclusion