Conserved lysin and arginin residues in the extracellular loop of P2X3 receptors are involved in agonist binding

Wolfgang Fischer, Z. Zádori, Yvonne Kullnick, Helke Gröger-Arndt, Heike Franke, Kerstin Wirkner, Peter Illes, Peter P. Mager

Research output: Contribution to journalArticle

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Abstract

Wild-type human (h) P2X3 receptors expressed in HEK293 cells responded to the prototypic agonist α,β-methylene ATP (α,β-meATP) with rapidly desensitizing inward currents and an increase in the intracellular Ca2+ concentration. In contrast to electrophysiological recordings, Ca2+ microfluorimetry showed a lower maximum of the concentration-response curve of α,β-meATP in the transiently than in the permanently transfected HEK293 cells. However, the concentrations causing 50% of the maximum possible effect (EC50 values) were identical, when measured with either method. In order to determine the role of certain conserved, positively charged amino acids in the nucleotide binding domains (NBD-1-4) of hP2X3 receptors for agonist binding, the lysine-63, -65, -176 and -299 as well as the arginine-281 and -295 residues were substituted by the neutral amino acid alanine. We observed no effect of α,β-meATP at the K63A, K176A, R295A, and K299A mutants, and a marked decrease of agonist potency at the K65A and R281A mutants. The P2X3 receptor antagonist 2',3'-O-trinitrophenyl-ATP (TNP-ATP) blocked the effect of α,β-meATP at the wild-type hP2X3 receptor with lower affinity than at the mutant K65A, indicating an interference of this mutation with the docking of the antagonist with its binding sites. The use of confocal fluorescence microscopy in conjunction with an antibody raised against the extracellular loop of the hP2X3 receptor documented the expression of all mutants in the plasma membrane of HEK293 cells. Eventually, we modelled the possible agonist and antagonist binding sites NBD-1-4 of the hP2X3 subunit by using structural bioinformatics. This model is in complete agreement with the available data and integrates results from mutagenesis studies with geometry optimization of the tertiary structure predictions of the receptor.

Original languageEnglish
Pages (from-to)7-17
Number of pages11
JournalEuropean Journal of Pharmacology
Volume576
Issue number1-3
DOIs
Publication statusPublished - Dec 8 2007

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Purinergic P2X3 Receptors
HEK293 Cells
Binding Sites
Cytophotometry
Neutral Amino Acids
Computational Biology
Fluorescence Microscopy
Confocal Microscopy
Mutagenesis
Alanine
Lysine
Arginine
Nucleotides
Adenosine Triphosphate
Cell Membrane
Amino Acids
Mutation
Antibodies

Keywords

  • Agonist binding
  • Antagonist binding
  • ATP
  • P2X receptor
  • Site directed mutagenesis

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Pharmacology

Cite this

Conserved lysin and arginin residues in the extracellular loop of P2X3 receptors are involved in agonist binding. / Fischer, Wolfgang; Zádori, Z.; Kullnick, Yvonne; Gröger-Arndt, Helke; Franke, Heike; Wirkner, Kerstin; Illes, Peter; Mager, Peter P.

In: European Journal of Pharmacology, Vol. 576, No. 1-3, 08.12.2007, p. 7-17.

Research output: Contribution to journalArticle

Fischer, Wolfgang ; Zádori, Z. ; Kullnick, Yvonne ; Gröger-Arndt, Helke ; Franke, Heike ; Wirkner, Kerstin ; Illes, Peter ; Mager, Peter P. / Conserved lysin and arginin residues in the extracellular loop of P2X3 receptors are involved in agonist binding. In: European Journal of Pharmacology. 2007 ; Vol. 576, No. 1-3. pp. 7-17.
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AU - Gröger-Arndt, Helke

AU - Franke, Heike

AU - Wirkner, Kerstin

AU - Illes, Peter

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