Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors. (December 2015)

• Record Type: Journal Article
• Authors: Kowalski, Maria; Hausmann, Ralf; Schmid, Julia; Dopychai, Anke; Stephan, Gabriele; Tang, Yong; Schmalzing, Günther; Illes, Peter; Rubini, Patrizia
• Is Part Of: Neuropharmacology. Volume 99(2015)
• Page Start: 115
• Page End: 130
• Publication Date: 2015-12
• Subjects: Heteromeric P2X receptors -- P2X2/3 receptors -- Subunit stoichiometry -- Mutagenesis; 2-MeSATP 2-methylthio ATP -- ASIC acid-sensing ion channel -- BN-PAGE blue native PAGE -- DRG dorsal root ganglion -- EC50 half maximal effective concentration -- FR fluorescent ratio -- GFP green fluorescent protein -- Imax peak current caused by maximum agonist concentration -- LGIC ligand-gated ion channel -- nH Hill coefficient -- TNP-ATP trinitrophenyl-ATP -- wt wild type -- α, β-meATP α, β-methylene ATP; Neuropsychopharmacology -- Periodicals; Autonomic Agents -- Periodicals; Neuropsychopharmacologie -- Périodiques; Neuropsychopharmacology; Periodicals; Electronic journals; 615.78
• DOI: 10.1016/j.neuropharm.2015.07.008
• Languages: English
• ISSNs: 0028-3908

Abstract

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 Zn 2+ 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. Highlights: Two distinct heteromeric complexes of P2X2/3 receptors occur in expression systems. The stoichiometry of P2X2/3 receptors depends on the relative amount of their subunits. The antagonist sensitivity of P2X2/3 receptors depends on their subunit stoichiometry. Zn 2+ and H + act at P2X2/3 receptors independent of their subunit stoichiometry.