1. Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors
- Author
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Anke Dopychai, Peter Illes, Maria Kowalski, Günther Schmalzing, Julia Schmid, Gabriele Stephan, Yong Tang, Patrizia Rubini, and Ralf Hausmann
- Subjects
Patch-Clamp Techniques ,Purinergic P2X Receptor Antagonists ,Cations, Divalent ,Protein subunit ,Allosteric regulation ,Gi alpha subunit ,Biology ,Transfection ,Membrane Potentials ,Interleukin 10 receptor, alpha subunit ,Purinergic P2X Receptor Agonists ,Xenopus laevis ,Cellular and Molecular Neuroscience ,Adenosine Triphosphate ,Allosteric Regulation ,Phenols ,Heterotrimeric G protein ,Animals ,Humans ,Polycyclic Compounds ,Receptor ,Pharmacology ,Dose-Response Relationship, Drug ,Cell Membrane ,Wild type ,body regions ,HEK293 Cells ,Biochemistry ,Competitive antagonist ,Mutation ,Oocytes ,Calcium ,Receptors, Purinergic P2X3 ,Receptors, Purinergic P2X2 - 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.
- Published
- 2015