Abstract

P2X receptors are a family of ion channels gated by extracellular ATP. Each member of the family can form functional homomeric channels, but only P2X₂ and P2X₃ have been shown to combine to form a unique heteromeric channel. Data from in situhybridization studies suggest that P2X₁ and P2X₅ may also co-assemble. In this study, we tested this hypothesis by expressing recombinant P2X₁ and P2X₅ receptor subunits either individually or together in human embryonic kidney 293 cells. In cells expressing the homomeric P2X₁ receptor, 30 μm α,β-methylene ATP (α,β-me-ATP) evoked robust currents that completely desensitized in less than 1 sec, whereas α,β-me-ATP failed to evoke current in cells expressing the homomeric P2X₅ receptor. By contrast, α,β-me-ATP evoked biphasic currents with a pronounced nondesensitizing plateau phase in cells that co-expressed both subunits. Further, the EC₅₀ for α,β-me-ATP was greater in cells expressing both P2X₁ and P2X₅ than in cells expressing P2X₁ alone (5 and 1.6 μm, respectively). Heteromeric assembly was confirmed using a co-immunoprecipitation assay of epitope-tagged P2X₁ and P2X₅ subunits. In summary, this study provides biochemical and functional evidence of a novel channel formed by P2X subunit heteropolymerization. This finding suggests that heteromeric subunit assembly constitutes an important mechanism for generating functional diversity of ATP-mediated responses.