Evidence for Functional P2X4/P2X7 Heteromeric Receptors

The cytolytic ionotropic ATP receptor P2X 7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X 7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X 4 , they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X 4 and P2X 7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X 4 and P2X 7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X 4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X 7 although there was no change in overall expression. Coexpression of a dominant-negative P2X 4 mutant (C353W) with P2X 7 , inhibited P2X 7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non–dominant-negative mutant (S341W) did not. Coexpression of P2X 4 S341W with P2X 7 produced a current that was potentiated by ivermectin and inhibited by 2′,3′- O -(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X 7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X 4 and P2X 7 , which suggests that they associate to form heteromeric receptors.