Biochemical and functional evidence for heteromeric assembly of P2X ₁ and P2X₄ subunits

P2X receptors are ligand-gated ion channels activated by extracellular ATP. In expression systems, P2X subunits form homo- and heterotrimeric receptors. Heteromerization is also likely to occur in vivo as (i) most P2X subunits show overlapping distribution in different tissues and (ii) the functional properties of many native P2X receptors differ from those of heterologously expressed homomeric receptors. Here, we used the Xenopus laevis oocyte expression system to test for heteromerization of P2X₁ and P2X₄ subunits. Upon co-injection, P2X₄ subunits were co-purified with hexahistidyl-tagged P2X₁ subunits indicating heteromerization. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis of these P2X complexes excluded artificial aggregation and confirmed that both subunits were present in trimeric complexes of the same size. Two-electrode voltage-clamp experiments revealed functional P2X receptors with kinetic properties resembling homomeric P2X₄ receptors and a pharmacological profile similar to homomeric P2X₁ receptors. Thus, application of α,β- methylene ATP evoked a slowly desensitizing current sensitive to the antagonists suramin and 2′,3′-O(2,4,6-trinitrophenyl)-ATP. This study provides for the first time biochemical and functional evidence for the formation of heteromeric P2X₁₊₄ receptors. These receptors may account for native P2X mediated responses that until now could not be correlated with previously described recombinant P2X receptors.