Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors

NEUROPHARMACOLOGY

卷:99

Article

Kowalski, Maria¹  Hausmann, Ralf²  Schmid, Julia¹  Dopychai, Anke²  Stephan, Gabriele¹  Tang, Yong³  Schmalzing, Guenther²  Illes, Peter¹  Rubini, Patrizia¹ 

[1] Univ Leipzig, Rudolf Boehm Inst Pharmacol & Toxicol, D-04107 Leipzig, Germany

[2] Rhein Westfal TH Aachen, Mol Pharmacol, D-52074 Aachen, Germany

[3] Chengdu Univ Tradit Chinese Med, Acupuncture & Tuina Sch, Chengdu 610075, Peoples R China

关键词: Heteromeric P2X receptors;    P2X2/3 receptors;    Subunit stoichiometry;    Mutagenesis;   

DOI  :  10.1016/j.neuropharm.2015.07.008

来源: Elsevier

【 摘 要 】

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 alpha,beta-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 alpha,beta-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 alpha,beta-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. (C) 2015 Elsevier Ltd. All rights reserved.

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