ATP Binding Site Mutagenesis Reveals Different Subunit Stoichiometry of Functional P2X2/3 and P2X2/6 Receptors

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 Ca2+ transients by the Ca2+-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. Background: Heteromeric P2X receptors increase the diversity of rapid ATP signaling. Results: Non-functional P2X2, P2X3, and P2X6 subunit mutants were used to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. Conclusion: The subunit stoichiometry of P2X2/3 and P2X2/6 is 1:2 and 2:1, respectively. Significance: Recognition sites between P2X2 and its partners rather than random association may govern the subunit composition of the receptor trimers.