The P2X 7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X 7 receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X 7 function was measured by ATPinduced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into various lymphocyte subsets and was decreased to values of ϳ25% of normal. The expression of the P2X 7 receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X 7 carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-␥ up-regulated P2X 7 function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X 7 receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.The purinergic P2X 7 receptor is a ligand-gated channel, selective for cationic permeants, which has a wide distribution including cells of the immune and hemopoietic system (1, 2). Activation of this receptor by brief exposure to extracellular ATP opens a channel that allows Ca 2ϩ and Na ϩ influx and K ϩ efflux and that initiates a cascade of intracellular downstream events. These include the stimulation of phospholipase D (3, 4), the activation of membrane metalloproteases (5-7), and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell (8, 9). P2X 7 activation also leads to extensive membrane blebbing (10), which is a typical morphological feature of the apoptotic process. P2X 7 receptors have two transmembrane domains with intracellular amino and carboxyl termini, and the P2X 7 receptor differs from other members of the P2X receptor family in having a long carboxyl terminus of 240 amino acids from the inner membrane face (11). This long carboxyl terminus is necessary for the permeability properties of the P2X 7 receptor because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12). P2X 7 has an oligomeric structure in the membrane based on trimeric or larger complexes of identical subunits (13,14), and there is evidence that P2X 7 interacts with a number of structural and adhesion proteins in a complex at the cell surface (15). Phosphorylation of a tyrosine at amino acid 343 of the P2X 7 primary structure has been proposed as being important for maintaining the full activity of the P2X 7 channel (15). A number of regulatory d...
The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.
The P2X 7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X 7 receptor have been observed, explained in part by loss-of-function polymorphisms that change Glu 496 to Ala (E496A) and Ile 568 to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg 307 (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X 7 function was measured by ATP-induced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X 7 carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X 7 and blocks P2X 7 function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X 7 function in cells heterozygous for the R307Q to a value 10 -40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X 7 , and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X 7 receptor, and since the affected Arg 307 is homologous to those amino acids essential for ATP binding to P2X 1 and P2X 2 , it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X 7 .In cells of the hemopoietic and immune systems, extracellular ATP can induce cytolysis of lymphocytes (1), monocytes/ macrophages (2), and dendritic cells (3). It is generally accepted that these cytolytic effects of ATP are mediated by the P2X 7 receptor, which is a ligand-gated cation channel activated by extracellular ATP and highly expressed on these cell types (4, 5 P2X receptors have an oligomeric structure in the plasma membrane based on trimeric or larger complexes of identical subunits (20,21). Moreover the values of Hill coefficients derived from the sigmoid ATP dose-response curves of the P2X 7 (P2Z) receptor are consistent with multiple ATP-binding sites in each P2X 7 trimer (8,22,23). All seven members of the P2X receptor family have two transmembrane domains with intracellular amino and carboxyl termini. Little is known of the conformation of the extracellular domain containing the ATPbinding site(s). An analysis of the P2X subtype sequence homology has shown that the two transmembrane domains M1 and M2 are separated by an extracellular sequence containing a cysteine-rich region (residues 110 -170) followed by a segment from Phe 188 -Val 321 that may form six antiparallel -pleated sheets homologous with members of the class II aminoacyl-tRNA synthetases (24). The ATP-...
Priming of monocytes with LPS produces large quantities of intracellular, biologically inactive IL-1β that can be processed and released by subsequent activation of the P2X7 receptor by extracellular ATP. We examined whether a loss-of-function polymorphism of the human P2X7 receptor (Glu496Ala) impairs this process. Both ATP-induced ethidium+ uptake and ATP-induced shedding of L-selectin (CD62L) were nearly absent in monocytes from four subjects homozygous for Glu496Ala confirming that this polymorphism impairs P2X7 function. The level of ATP-induced IL-1β released in 2 h from LPS-activated whole blood from homozygous subjects was 50% of that from wild-type samples. A more marked defect in IL-1β release was observed from LPS-activated monocytes of homozygous subjects which was only 22% of that released from wild-type monocytes after a 30-min incubation with ATP. However, after a 60-min incubation with ATP, the amount of IL-1β released from homozygous monocytes was 70% of that released from wild-type monocytes. Incubation of monocytes of either genotype with nigericin resulted in a similar release of IL-1β. Western blotting demonstrated that ATP induced the release of mature 17-kDa IL-1β from monocytes, and confirmed that this process was impaired in homozygous monocytes. Finally, ATP-induced 86Rb+ efflux was 9-fold lower from homozygous monocytes than from wild-type monocytes. The results indicate that ATP-induced release of IL-1β is slower in monocytes from subjects homozygous for the Glu496Ala polymorphism in the P2X7 receptor and that this reduced rate of IL-1β release is associated with a lower ATP-induced K+ efflux.
SummaryPhospholipase D (PLD) hydrolyzes the phosphodiester bond of the predominant membrane phospholipid, phosphatidylcholine producing phosphatidic acid and free choline. This activity can participate in signal transduction pathways and impact on vesicle trafficking for secretion and endocytosis, as well as receptor signalling. Phospholipids can regulate PLD activity directly, through specific intermolecular interactions, or indirectly, through their effect on the localization or activity of PLD's protein effectors. This short review highlights these various phospholipid inputs into the regulation of PLD activity and also reviews potential roles for PLD-generated phosphatidic acid, particularly a mechanism by which the phospholipid may participate in the process of vesicular trafficking.
Canine erythrocytes are known to undergo a reversible increase in cation permeability when incubated with extracellular ATP. We have examined the expression and function of P2X receptors on human erythrocytes using confocal microscopy and a panel of anti-P2X 1-7 antibodies and have measured monovalent cation fluxes in the presence of various nucleotide agonists. Human erythrocytes expressed P2X 7 receptors on all cells examined from eight of eight subjects, as well as P2X 2 at a far lower staining intensity in six of eight subjects. ATP stimulated the efflux of 86 Rb ؉ (K ؉ ) from human erythrocytes in a dose-dependent fashion with an EC 50 of ϳ95 M. Other nucleotides also induced an efflux of 86 Rb ؉ from erythrocytes with an order of agonist potency of 2 -and 3 -O(4-benzoylbenzoyl) ATP (BzATP) > ATP > 2-methylthio-ATP (2MeSATP) > adenosine 5 -O-(3-thiotriphosphate) (ATP␥S), whereas ADP or UTP had no effect. ATP-induced efflux of 86 Rb ؉ from erythrocytes was inhibited by extracellular Na ؉ and oxidized ATP, as well as by KN-62, an antagonist specific for the human P2X 7 receptor. When erythrocytes were incubated in isotonic KCl medium, the addition of ATP stimulated an 86 Rb ؉ influx approximately equal in magnitude to ATPstimulated 86 Rb ؉ efflux from the same cells. BzATP also stimulated the influx of 22 Na ؉ into erythrocytes incubated in isotonic NaCl medium. Both ATP-induced efflux and influx of 86 Rb ؉ and 22 Na ؉ were impaired in erythrocytes from subjects who had inherited loss-offunction polymorphisms in the P2X 7 receptor. These results suggest that the reversible permeabilization of erythrocytes by extracellular ATP is mediated by the P2X 7 receptor.Many of our concepts of cellular Na ϩ and K ϩ homeostasis were based on experiments in the erythrocyte, a cell type in which intracellular Na ϩ and K ϩ concentrations could be readily changed and the subsequent effect on ion transport could be measured. Early studies of the sodium pump used the technique of hypotonic hemolysis and resealing of ghosts to study the dependence of Na ϩ pumping on intracellular cation concentration (1, 2), whereas the first description of Na ϩ -K ϩ -2Cl Ϫ cotransport owed much to a nystatin technique to alter the Na ϩ gradient and change the driving force for cotransport (3). Yet another technique was introduced by Parker et al. (4,5) who showed that intracellular Na ϩ and K ϩ concentration of canine erythrocytes could be equilibrated with the cation concentration in the medium simply by incubation with extracellular ATP for 30 -60 min and that removal of ATP restored the basal permeability of the cell. This reversible effect of ATP suggested the involvement of an ATP-gated cation channel, but it is only with recent observations and knowledge of the P2X receptor family (6) that it is possible to study this question.Seven subtypes of the P2X receptor family have been identified based on a common structure of two transmembrane domains with intracellular amino and carboxyl termini and a trimeric structure in the plasma membrane ...
Over three decades ago, Parker and Snow (Am J Physiol 223: 888-893, 1972) demonstrated that canine erythrocytes undergo an increase in cation permeability when incubated with extracellular ATP. In this study we examined the expression and function of the channel/pore-forming P2X(7) receptor on canine erythrocytes. P2X(7) receptors were detected on canine erythrocytes by immunocytochemistry and immunoblotting. Extracellular ATP induced (86)Rb(+) (K(+)) efflux from canine erythrocytes that was 20 times greater than that from human erythrocytes. The P2X(7) agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-trisphosphate (BzATP) was more potent than ATP, and both stimulated (86)Rb(+) efflux from erythrocytes in a dose-dependent fashion with EC(50) values of approximately 7 and approximately 309 microM, respectively. 2-Methylthioadenosine 5'-triphosphate and adenosine 5'-O-(3-thiotriphosphate) induced a smaller (86)Rb(+) efflux from erythrocytes, whereas ADP, AMP, UTP, or adenosine had no effect. ATP-induced (86)Rb(+) efflux from erythrocytes was inhibited by oxidized ATP, KN-62, and Brilliant blue G, known P2X(7) antagonists. ATP also induced uptake of choline(+) into canine erythrocytes that was 60 times greater than that into human erythrocytes. Overnight incubation of canine erythrocytes with ATP and BzATP induced phosphatidylserine exposure in >80% of cells and caused up to 20% hemolysis. In contrast, <30% of human erythrocytes showed phosphatidylserine exposure after overnight incubation with ATP and BzATP, and hemolysis was negligible. Flow cytometric measurements of ATP-induced ethidium(+) uptake showed that P2X(7) function was three times lower in canine monocytes than in human monocytes. These data show that the massive cation permeability increase induced by extracellular ATP in canine erythrocytes results from activation and opening of the P2X(7) receptor channel/pore.
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