No abstract

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.

The human P2X7 receptor is a two-transmembrane ionotropic receptor which has a ubiquitous distribution and is most highly expressed on immune cells. In macrophages and similar myeloid cells primed by lipopolysaccharide (LPS), activation of P2X7 by extracellular ATP opens a cation channel/pore allowing massive K+ efflux associated with processing and secretion of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. A variety of other downstream effects follows P2X7 activation over several minutes including shedding of certain surface molecules, membrane blebbing, microvesicle/exosome release and apoptosis of the cell. High concentrations of ATP (>100 µM) are required to activate P2X7 but it remains unclear where these levels exist, other than in inflammatory foci or confined spaces such as in bone. A variety of potent selective antagonists of P2X7 activation have recently become available, allowing clinical trials to be undertaken in inflammatory and immune-mediated disorders. Proteomic studies have shown that P2X7 exists as a large multiprotein complex which includes non-muscle myosin heavy chain and other elements of the cytoskeleton. In the absence of its ATP ligand and serum, P2X7 has an alternate function in the recognition and phagocytosis of non-opsonized foreign particles, including bacteria and apoptotic cells. The P2RX7 gene has many polymorphic variants and isoforms which increase or decrease function of the receptor. Genetic association studies have linked loss-of-function polymorphisms with reactivation of latent tuberculosis as well as symptomatic infection with certain other obligate intracellular pathogens. The many roles involving P2X7 suggest that this receptor is essential to fundamental aspects of the innate immune response.

Adenosine 5′-triphosphate is a well-known extracellular signaling molecule and neurotransmitter known to activate purinergic P2X receptors. Information has been elucidated about the structure and gating of P2X channels following the determination of the crystal structure of P2X4 (zebrafish), however, there is still much to discover regarding the role of this receptor in the central nervous system (CNS). In this review we provide an overview of what is known about P2X4 expression in the CNS and discuss evidence for pathophysiological roles in neuroinflammation and neuropathic pain. Recent advances in the development of pharmacological tools including selective antagonists (5-BDBD, PSB-12062, BX430) and positive modulators (ivermectin, avermectins, divalent cations) of P2X4 will be discussed.

The human P2X7 receptor is a trimeric ligand-gated cation channel coded by the P2XR7 gene located at chromosome position 12q24. P2X7 is expressed in a wide variety of normal and disease-associated cell types. Activation of this receptor by extracellular adenosine 5'-triphosphate results in numerous downstream events including the release of pro-inflammatory mediators, cell proliferation or death, and killing of intracellular pathogens. As a result, P2X7 plays important roles in inflammation, immunity, bone homeostasis, neurological function and neoplasia. The P2XR7 gene encodes a P2X7 subunit 595 amino acids in length, however splice isoforms that can alter receptor expression and function, and modify the signaling properties downstream of receptor activation also exist. Moreover, the relative amount of P2X7 function varies between human individuals due to numerous single nucleotide polymorphisms resulting in either loss- or gain-of-function. Combinations of these polymorphisms give rise to various haplotypes that can also modify P2X7 function. Collectively, P2X7, and its splice and polymorphic variants are attracting considerable interest in relation to human health and disease, including the development and publication of a number of patents.

No abstract

The P2RX7 gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) underlie the wide variation observed in P2X7 receptor responses. We review the discovery of those non-synonymous SNPs that affect receptor function and compare their frequencies in different ethnic populations. Analysis of pairwise linkage disequilibrium (LD) predicts a limited range of haplotypes. The strong LD between certain functional SNPs provides insight into published studies of the association between SNPs and human disease.

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X(7) channel and massive K(+) efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X(7) were isolated by using anti-P2X(7) monoclonal antibody-coated Dynabeads from both interferon-gamma plus LPS-stimulated monocytic THP-1 cells and P2X(7)-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X(7) in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X(7) receptor by ATP caused dissociation of P2X(7) from nonmuscle myosin in both cell types. The interaction of P2X(7) and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X(7) pore function without any increase in surface expression or ion channel function of P2X(7) receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X(7)-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X(7) and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X(7) channel to a pore.