title: ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors.
abstract: The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.
authors.affiliation: Department of Molecular Pharmacology, University Hospital of Rheinisch Westfaelische Technische Hochschule, Aachen University, 52074 Aachen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hausmann
authors.forename: Mandy
authors.initials: M
authors.lastname: Bodnar
authors.forename: Ronja
authors.initials: R
authors.lastname: Woltersdorf
authors.forename: Haihong
authors.initials: H
authors.lastname: Wang
authors.forename: Martin
authors.initials: M
authors.lastname: Fuchs
authors.forename: Nanette
authors.initials: N
authors.lastname: Messemer
authors.forename: Ying
authors.initials: Y
authors.lastname: Qin
authors.forename: Janka
authors.initials: J
authors.lastname: Günther
authors.forename: Thomas
authors.initials: T
authors.lastname: Riedel
authors.forename: Marcus
authors.initials: M
authors.lastname: Grohmann
authors.forename: Karen
authors.initials: K
authors.lastname: Nieber
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
authors.forename: Patrizia
authors.initials: P
authors.lastname: Rubini
authors.forename: Peter
authors.initials: P
authors.lastname: Illes
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D019281:Dimerization; D004594:Electrophysiology / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D018048:Receptors, Purinergic P2 / Q000737:chemistry*; D058476:Receptors, Purinergic P2X2 / Q000737:chemistry*; D058477:Receptors, Purinergic P2X3 / Q000737:chemistry*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013499:Surface Properties; D014982:Xenopus laevis
chemical_list: D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; C110590:purinoceptor P2X6; D000255:Adenosine Triphosphate; D002118:Calcium