title: Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors.
abstract: The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wild type P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration-response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 receptors. Whereas the allosteric modulators H+ and Zn2+ failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of P2X2 and P2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP)2/(P2X3)1 complex and a (P2X2-GFP)1/(P2X3)2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits.
authors.affiliation: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Kowalski
authors.affiliation: Molecular Pharmacology, RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hausmann
authors.affiliation: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Schmid
authors.affiliation: Molecular Pharmacology, RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Dopychai
authors.affiliation: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Stephan
authors.affiliation: Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, 610075 Chengdu, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Molecular Pharmacology, RWTH Aachen University, 52074 Aachen, Germany.
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
authors.affiliation: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Illes
authors.affiliation: Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany. Electronic address: Patrizia.Rubini@medizin.uni-leipzig.de.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Rubini
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009154:Mutation; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010636:Phenols / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000494:pharmacology; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D058476:Receptors, Purinergic P2X2 / Q000235:genetics / Q000378:metabolism*; D058477:Receptors, Purinergic P2X3 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014982:Xenopus laevis
chemical_list: C470346:A-317491; D002413:Cations, Divalent; C546401:P2RX2 protein, human; C546407:P2RX3 protein, human; D010636:Phenols; D011083:Polycyclic Compounds; D058911:Purinergic P2X Receptor Agonists; D058920:Purinergic P2X Receptor Antagonists; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium
keywords: Heteromeric P2X receptors; Mutagenesis; P2X2/3 receptors; Subunit stoichiometry