Tags

Type your tag names separated by a space and hit enter

Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors.
Neuropharmacology 2015; 99:115-30N

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+Show Affiliations

Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.Molecular Pharmacology, RWTH Aachen University, 52074 Aachen, Germany.Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.Molecular Pharmacology, RWTH Aachen University, 52074 Aachen, Germany.Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, 610075 Chengdu, China.Molecular Pharmacology, RWTH Aachen University, 52074 Aachen, Germany.Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany.Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany. Electronic address: Patrizia.Rubini@medizin.uni-leipzig.de.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

26184350

Citation

Kowalski, Maria, et al. "Flexible Subunit Stoichiometry of Functional Human P2X2/3 Heteromeric Receptors." Neuropharmacology, vol. 99, 2015, pp. 115-30.
Kowalski M, Hausmann R, Schmid J, et al. Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors. Neuropharmacology. 2015;99:115-30.
Kowalski, M., Hausmann, R., Schmid, J., Dopychai, A., Stephan, G., Tang, Y., ... Rubini, P. (2015). Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors. Neuropharmacology, 99, pp. 115-30. doi:10.1016/j.neuropharm.2015.07.008.
Kowalski M, et al. Flexible Subunit Stoichiometry of Functional Human P2X2/3 Heteromeric Receptors. Neuropharmacology. 2015;99:115-30. PubMed PMID: 26184350.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors. AU - Kowalski,Maria, AU - Hausmann,Ralf, AU - Schmid,Julia, AU - Dopychai,Anke, AU - Stephan,Gabriele, AU - Tang,Yong, AU - Schmalzing,Günther, AU - Illes,Peter, AU - Rubini,Patrizia, Y1 - 2015/07/13/ PY - 2015/02/13/received PY - 2015/07/01/revised PY - 2015/07/09/accepted PY - 2015/7/18/entrez PY - 2015/7/18/pubmed PY - 2016/8/25/medline KW - Heteromeric P2X receptors KW - Mutagenesis KW - P2X2/3 receptors KW - Subunit stoichiometry SP - 115 EP - 30 JF - Neuropharmacology JO - Neuropharmacology VL - 99 N2 - 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. SN - 1873-7064 UR - https://www.unboundmedicine.com/medline/citation/26184350/Flexible_subunit_stoichiometry_of_functional_human_P2X2/3_heteromeric_receptors_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0028-3908(15)30014-9 DB - PRIME DP - Unbound Medicine ER -