Binding site composition and subunit stoichiometry of ATP-sensitive homomeric P2X3 andheteromeric P2X2/3 receptors

Homomeric P2X3 and heteromeric P2X2/3 receptors (Rs) are present in sensory ganglia and participate in pain sensation. In a search for the ATP binding pouch of the P2X3R we replaced amino acid (AA) residues in the four supposed nucleotide binding segments (NBSs) of the human P2X3R by alanine and these mutants were expressed in HEK293 cells. Patch clamp measurements as well as Ca2+ imaging techniques were used to compare the concentration-response curves of the selective P2X1,3R agonist α,β-methylene ATP (α,β-meATP) obtained at the wild-type P2X3R and its NBS mutants. Within these NBSs, certain adjacent AAs were of great importance for a full agonist response. We concluded that groups of AAs organized in NBSs rather than individual AAs appear to be responsible for agonist binding at the P2X3R. These NBSs are located at the interface ofthe three subunits forming a functional receptor. The aim of the following experiments was to clarify the subunit stoichiometry of P2X2/3Rs, where two identical subunits forma receptor with a different partner. For this purpose, four non-functional Ala mutants of the P2X2and P2X3 subunits were generated by replacing single, homologous AAs particularly important for agonist binding. Co-expression of these mutants in HEK293 cells (1:2 or 4:1 transfection ratios for P2X2/3) to yield P2X2-wt/P2X3- mutant or P2X2-mutant/P2X3-wt receptors, the abolition of agonist responses depended on the transfection ratios. The effects of α,β-meATP and 2-methylthio ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca2+ transients by the Ca2+ imaging method. We conclude that with P2X2/3Rs the subunit composition depends on the transfection ratios. In case of a 1:2 ratio of P2X2- and P2X3-cDNAs both (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 variants are generated with a predominance of the former one, whereas in case of a 4:1 ratio of these cDNAs the subunit stoichiometry isreversed. It is suggested that both P2X2/3 receptor variants may be present in DRG neurons with possibly different sensitivities to agonists and antagonists

کلیدواژه ها:

Stoichiometry ، P2X2/3 receptors ، P2X3 receptors