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

Abstract

ATP-gated P2X 7 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. P2X 7 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 P2X 7 subunit makes. We constructed and purified an ectodomain protein of the rat P2X 7 receptor (amino acids 60 -323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X 7 receptors in central and peripheral immune cells. In HEK cells expressing rat P2X 7 receptors, the Ab increased the maximum current evoked by BzATP by 3-8-fold with a 5-fold leftward shift in EC 50 concentration. This Ab recognized only a nondenatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X 7 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 P2X 7 receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X 7 receptors in brain glia and/or astrocytes appear to form only as monomeric subunits.Like most neurotransmitters, extracellular ATP activates both metabotropic (G protein-coupled) receptors, the P2Y receptor family, and ionotropic (ligand-gated) receptors, the P2X receptor family (1). When compared with other ionotropic receptors (e.g. nicotinic), which are primarily or solely expressed in nerve and muscle, P2X receptors are unusual in their wide spread expression in both excitable and non-excitable cells (1-3). There are seven P2X receptors, six of which (P2X 1-6 ) are found in brain and peripheral neurons; additionally, mRNA for P2X 1 , P2X 4 , and the non-neuronal P2X 7 receptor are prominent in many immune cells, particularly monocytes, macrophage, bone marrow, and brain microglia (1-3).The P2X 7 receptor shows 40 -45% amino acid identity with any one of the other P2X receptors; it shares the overall membrane topology of two transmembrane domains, intracellular N and C termini, and the large ectodomain with conservation of the 10 extracellular cysteine residues, although its C-terminal is some 200 amino acids longer than other P2X receptors (1-3). However, the functional sequelae of P2X 7 receptor activation differ strikingly from other P2X receptors. Activation of P2X 7 receptors expressed in mammalian cells, such as HEK 1 or CHO cells, opens both a small cationic channel and a large (up to 900 Da) dye-permeable pore, followed within seconds by extensive membrane blebbing (4, 5); similar events occur upon activation of native P2X 7 receptors in some but not all immune cells expressing this receptor (6 -8). The mechanism(s) underlying formation of the permeabilizing pore and other downstream events such as membrane blebbing are not known but the intracellular ...

Regulation of P2X7 gene transcription

“…Third, cells produce non-functional variants of the P2X 7 receptor [11,74,75] that can oligomerize with the functional fulllength receptor [11]. In epithelial cells, P2X 7 -mediated apoptosis involves formation of pores that are composed of oligomers of P2X 7 molecules [11,76] and pannexins [77,78], but co-expression of variants, e.g., the P2X 7-j [11] would favor formation of non-active complexes of the receptor [11]. Together, these data indicate that under physiological conditions, the P2X 7 mechanism is tightly controlled and regulated.…”

Purinergic receptors expressed in human skeletal muscle fibres

“…The following primary antibodies were used, either alone or two together (rabbit+mouse): Rabbit polyclonal anti-P2X 1 (APR-001, Alomone Labs, Israel; 1:500; previously used in the following studies [6,8,[13][14][15][16][17][18][19]); mouse polyclonal anti-P2X 4 (H00005025-B01P; Abnova, Taiwan; 1:500); rabbit polyclonal anti-P2Y 1 (APR-009, Alomone Labs; 1:500; previously used in the following studies [18,[20][21][22]); polyclonal anti-P2Y 2 (APR-010, Alomone Labs; 1:500; previously used in the following studies [6,8,18,[22][23][24]); rabbit polyclonal anti-P2Y 4 (P6497, Sigma-Aldrich, Denmark; 1:250); rabbit polyclonal anti-P2Y 11 (APR-015, Alomone Labs; 1:500; previously used in the following studies [18,[25][26][27][28][29][30]); rabbit polyclonal anti-P2Y 12 (HPA013796, Sigma-Aldrich; 1:30; previously used in the following study [31]). The P2X 1 antibody was highly specific and directed against the epitope corresponding to amino acid residues 382-399 of rat P2X 1 (human 15/18 residues identical); the P2Y 11 antibody was corresponding to amino acid residues 357-373 of human P2Y 11 ; western blots for anti-P2X 1 and anti-P2Y 11 are shown on the Alomone homepage.…”

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

“…Functional P2X7Rs are homotrimers [28] abundantly expressed in cells of immunological origin or function, in particular macrophages [43], mast cells [7], and microglia [49], as well as in some types of cancer [31,42]. The receptor exhibits low sensitivity to ATP, does not desensitize, and gradually develops permeability to organic cations, causing a sustained current growth accompanied by cell blebbing and death [1,12,32,45,50].…”