Abstract

The cytolytic ionotropic ATP receptor P2X₇ has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X₇ receptors are frequently coexpressed with another subtype of P2X receptor, P2X₄, 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 P2X₄ and P2X₇ 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 P2X₄ and P2X₇ receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X₄ receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X₇ although there was no change in overall expression. Coexpression of a dominant-negative P2X₄ mutant (C353W) with P2X₇, inhibited P2X₇ receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non–dominant-negative mutant (S341W) did not. Coexpression of P2X₄S341W with P2X₇ 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 P2X₇ 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 P2X₄ and P2X₇, which suggests that they associate to form heteromeric receptors.