Search

Extracellular nucleotides are important mediators of activation, triggering various responses through plasma membrane P2 and P1 receptors. P2 receptors are further subdivided into ionotropic P2X receptors and G protein-coupled P2Y receptors. P2X4 is an ATP-gated cation channel broadly expressed in most tissues of the body. Within the P2X family, P2X4 has a unique subcellular distribution, being preferentially localized in lysosomes. In these organelles, high ATP concentrations do not trigger P2X4 because of the low pH. However, when the pH increases to 7.4, P2X4 can be stimulated by intra-lysosomal ATP, which is in its active, tetra-anionic form. Elucidation of P2X4, P2X3 and P2X7 structures has shed some light on the functional differences between these purinergic receptors. The potential interaction between P2X4 and P2X7 has been extensively studied. Despite intensive effort, it has not been possible yet to determine whether P2X4 and P2X7 interact as heterotrimers or homotrimers at the plasma membrane. However, several publications have shown that functional interactions between P2X4 and P2X7 do occur. Importantly, these studies indicate that P2X4 potentiates P2X7-dependent activation of inflammasomes, leading to increased release of IL-1β and IL-18. The role of P2X4 in various diseases could be beneficial or deleterious even though the pathophysiological mechanisms involved are still poorly defined. However, in diseases whose physiopathology involves activation of the NLRP3 inflammasome, P2X4 was found to exacerbate severity of disease. The recent production of monoclonal antibodies specific for the human and mouse P2X4, some of which are endowed with agonist or antagonist properties, raises the possibility that they could be used therapeutically. Analysis of single nucleotide polymorphisms of the human P2RX4 gene has uncovered the association of P2RX4 gene variants with susceptibility to several human diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kanellopoulos, Almeida-da-Silva, Rüütel Boudinot and Ojcius.)

P2X purinergic receptors are extracellular ATP-gated ion channel receptors present on the cell plasma membrane. P2X receptors have been found in Metazoa, fungi, amoebas, and in plants. In mammals, P2X7 is expressed by a large number of cell types and is involved in inflammation and immunity. Remarkably, P2X7 does not desensitize as other P2X do, a feature linked to a "C-cysteine anchor" intra-cytoplasmic motif encoded by exon 11. Another specific feature of P2X7 is its C-terminal cytoplasmic ballast domain (exon 13) which contains a zinc (Zn) coordinating cysteine motif and a GDP-binding region. To determine the origin of P2X7, we analyzed and compared sequences and protein motifs of the C-terminal intra-cytoplasmic region across all main groups of Metazoa. We identified proteins with typical ballast domains, sharing a remarkably conserved Zn-coordinating cysteine motif. Apart from vertebrates, these ballast domains were not associated with a typical P2X architecture. These results strongly suggest that P2X7 resulted from the fusion of a P2X gene, highly similar to P2X4, with an exon encoding a ballast domain. Our work brings new evidence on the origin of the P2X7 purinergic receptor and identifies the Zn-coordinating cysteine domain as the fundamental feature of the ancient ballast fold., (Copyright © 2020 Rump, Smolander, Rüütel Boudinot, Kanellopoulos and Boudinot.)

Extracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation, and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterized the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4 high expressing eosinophils.