Differential Assembly of Rat Purinergic P2X7 Receptor in Immune Cells of the Brain and Periphery

ATP-gated P2X7 purinoceptors are found in most immune cells of the periphery and the brain where their activation leads to multiple downstream events such as cell permeabilization, apoptosis, and/or cytokine release. P2X7 receptors do not form heteromeric receptors with any of the other six P2X subunits, and it is not known what type of homomeric assemblies the P2X7 subunit makes. We constructed and purified an ectodomain protein of the rat P2X7 receptor (amino acids 60-323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X7 receptors in central and peripheral immune cells. In HEK cells expressing rat P2X7 receptors, the Ab increased the maximum current evoked by BzATP by 3-8-fold with a 5-fold leftward shift in EC50 concentration. This Ab recognized only a non-denatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X7 Ab recognized both monomeric subunits on SDS-PAGE and a multimeric complex on blue native-PAGE in this heterologous expression system. With Western blotting using these two Abs, native P2X7 receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X7 receptors in brain glia and/or astrocytes appear to form only as monomeric subunits.