Endogenous P2X7 Research Articles

Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors

P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside–compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells.SIGNIFICANCE STATEMENTGinsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket.

P2X7 from j774 murine macrophages acts as a scavenger receptor for bacteria but not yeast

We studied the effects of extracellular ATP and Ca2+ on uptake of bacteria (Staphylococcus aureus or Escherichia coli) and live yeast (Candida glabrata) by J774 macrophages to determine the role of endogenous P2X7 receptors in phagocytosis. Our findings show that phagocytosis of bio-particles coated with S. aureus or E. coli was blocked by ATP and the P2X7 receptor agonist BzATP, while yeast phagocytosis was not. A438079, an antagonist of P2X7 receptors, partially reverted the effects of ATP on bacterial phagocytosis. To determine if P2X7-mediated Ca2+ entry into macrophages was blocking the engulfment of bacteria, we measured phagocytic activity in the absence or presence of 2 mM extracellular Ca2+ with or without ATP. Ca2+, in the absence of ATP, was required for engulfment of E. coli and C. glabrata but not S. aureus. Adding ATP inhibited phagocytosis of S. aureus and E. coli regardless of Ca2+, suggesting that Ca2+ entry was not important for inhibiting phagocytosis. On the other hand, phagocytosis of normal or hyper-adherent C. glabrata mutants had an absolute requirement for extracellular Ca2+ due to yeast adhesion to macrophages mediated by Ca2+-dependent adhesion proteins. We conclude that unstimulated P2X7 from J774 cells act as scavenger receptor for the uptake of S. aureus and E. coli but not of yeast; Ca2+ entry via P2X7 receptors play no role in phagocytosis of S. aureus and E. coli; while the effect of Ca2+ on C. glabrata phagocytosis was mediated by the adhesins Epa1, Epa6 and Epa7.

Atorvastatin prevents ATP-driven invasiveness via P2X7 and EHBP1 signaling in PTEN-expressing prostate cancer cells

Epidemiological studies indicate that statins, cholesterol-lowering drugs, prevent aggressive prostate cancer and other types of cancer. Employing essentially non-prostate cell lines, we previously showed that statins rapidly downregulate nuclear levels of phosphorylated Akt via P2X7, a purinergic receptor recently implicated in invasive growth. Here, we present studies on phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-positive prostatic cells. We document an involvement of EH domain-binding protein 1 (EHBP1), previously associated with aggressive prostate cancer and insulin-stimulated trafficking and cell migration, in P2X7 signaling. We also show that EHBP1 is essential for an anti-invasive effect of atorvastatin. Furthermore, EHBP1 interacted with P-Rex1, a guanine nucleotide exchange factor previously implicated in invasive growth. Mevalonate did not prevent this anti-invasive effect of atorvastatin. These data indicate that atorvastatin modulates invasiveness via P2X7, EHBP1 and P-Rex1. Interestingly, the interaction between EHBP1 and P-Rex1 was not induced by extracellular adenosine triphosphate (ATP), the endogenous P2X7 ligand, and statins counteracted invasiveness stimulated by extracellular ATP. In support of these experimental data, a population-based genetic analysis showed that a loss of function allele in the P2X7 gene (rs3751143) associated with non-aggressive cancer, and the common allele with aggressive cancer. Our data indicate a novel signaling pathway that inhibits invasiveness and that is druggable. Statins may reduce the risk of aggressive prostate cancer via P2X7 and by counteracting invasive effects of extracellular ATP.

MiR-150 Promotes Human Breast Cancer Growth and Malignant Behavior by Targeting the Pro-Apoptotic Purinergic P2X7 Receptor

The P2X7 receptor regulates cell growth through mediation of apoptosis. Low level expression of P2X7 has been linked to cancer development because tumor cells harboring a defective P2X7 mechanism can escape P2X7 pro-apoptotic control. microRNAs (miRNAs) function as negative regulators of post-transcriptional gene expression, playing major roles in cellular differentiation, proliferation, and metastasis. In this study, we found that miR-150 was over-expressed in breast cancer cell lines and tissues. In these breast cancer cell lines, blocking the action of miR-150 with inhibitors leads to cell death, while ectopic expression of the miR-150 results in increased cell proliferation. We deploy a microRNA sponge strategy to inhibit miR-150 in vitro, and the result demonstrates that the 3′-untranslated region (3′UTR) of P2X7 receptor contains a highly conserved miR-150-binding motif and its direct interaction with miR-150 down-regulates endogenous P2X7 protein levels. Furthermore, our findings demonstrate that miR-150 over-expression promotes growth, clonogenicity and reduces apoptosis in breast cancer cells. Meanwhile, these findings can be decapitated in nude mice with breast cancer xenografts. Finally, these observations strengthen our working hypothesis that up-regulation of miR-150 in breast cancer is inversely associated with P2X7 receptor expression level. Together, these findings establish miR-150 as a novel regulator of P2X7 and a potential therapeutic target for breast cancer.

P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase-dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2'(3')-O-(4-Benzoylbenzoyl)-ATP. The P2X7 inhibitors BBG, oATP, and KN-62 prevented 2'(3')-O-(4-Benzoylbenzoyl)-ATP-induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1-100μM). High concentrations of ATP (1mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7-induced motor neuron death was dependent on neuronal nitric oxide synthase-mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well-established peroxynitrite/FAS death pathway in motor neurons.

The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

P2X7 receptors are involved not only in physiological functions but also in pathological brain processes. Although an increasing number of findings indicate that altered receptor expression has a causative role in neurodegenerative diseases and cancer, little is known about how expression of P2rx7 gene is controlled. Here we reported the first molecular and functional evidence that Specificity protein 1 (Sp1) transcription factor plays a pivotal role in the transcriptional regulation of P2X7 receptor. We delimited a minimal region in the murine P2rx7 promoter containing four SP1 sites, two of them being highly conserved in mammals. The functionality of these SP1 sites was confirmed by site-directed mutagenesis and Sp1 overexpression/down-regulation in neuroblastoma cells. Inhibition of Sp1-mediated transcriptional activation by mithramycin A reduced endogenous P2X7 receptor levels in primary cultures of cortical neurons and astrocytes. Using P2rx7-EGFP transgenic mice that express enhanced green fluorescent protein under the control of P2rx7 promoter, we found a high correlation between reporter expression and Sp1 levels in the brain, demonstrating that Sp1 is a key element in the transcriptional regulation of P2X7 receptor in the nervous system. Finally, we found that Sp1 mediates P2X7 receptor up-regulation in neuroblastoma cells cultured in the absence of serum, a condition that enhances chromatin accessibility and facilitates the exposure of SP1 binding sites.

Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor

P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca(2+) content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agar-infiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.

P2X7 Receptor Activates Multiple Selective Dye-Permeation Pathways in RAW 264.7 and Human Embryonic Kidney 293 Cells

P2X7 receptor has gained an increasing importance as a drug target. One important response to P2X7 receptor stimulation is the uptake of large molecular weight tracers into cells. However, mechanism for this response is not understood clearly, but it is generally believed that a nonselective large pore protein forms this P2X7 receptor-activated permeability pathway. We examined human embryonic kidney (HEK) 293 cells transfected with rat P2X7 receptors (HEK-rP2X7) and a macrophage derived cell line, RAW 264.7, that expresses an endogenous P2X7 receptor. We used confocal microscopy to investigate uptake of different types of dyes into these cells after ATP application. Stimulation of P2X7 receptors in HEK-rP2X7 cells activated two different dye uptake pathways. The first was permeable to the cationic fluorescent dyes YO-PRO-1 and TO-TO-1 but not to the anionic dyes lucifer yellow and calcein and did not require intracellular Ca2+ concentration ([Ca2+](i)) increase to be activated. The second pathway permeated only lucifer yellow and was completely dependent on [Ca2+](i) for activation. In RAW 264.7 cells, P2X7 receptor stimulation activated uptake of ethidium, YO-PRO-1, TO-TO-1, lucifer yellow, and calcein. Again, two different permeation pathways were discerned in RAW 264.7 cells: one permeated only ethidium and the other one, only lucifer yellow. We did observed no clear [Ca2+](i) dependence for these permeation pathways. Our results demonstrate that instead of a single nonselective pore, P2X7 receptor seems to activate at least two permeation pathways, one for cationic and one for anionic dyes with different activation properties.

Peripheral and Central P2X3 Receptor Contributions to Colon Mechanosensitivity and Hypersensitivity in the Mouse

Irritable bowel syndrome is characterized by altered sensory qualities, namely discomfort/pain and colorectal hypersensitivity. In mice, we examined the role of P2X(3) receptors in colon mechanosensitivity and intracolonic zymosan-produced hypersensitivity, a model of persistent colon hypersensitivity without colon inflammation. The visceromotor response to colon distension (15-60 mm Hg) was determined before and after intracolonic saline or zymosan (30 mg/mL, 0.1 mL, daily for 3 days) treatment. Colon pathology and intracolonic adenosine triphosphate release was assessed in parallel experiments. To examine P2X(3) receptor contributions to colon mechanosensation and hypersensitivity, electrophysiologic experiments were performed using an in vitro colon-pelvic nerve preparation. Visceromotor responses to distension were significantly reduced in P2X(3)(+/-) and P2X(3)(-/-) mice relative to wild-type mice. Colon hypersensitivity produced by zymosan was virtually absent in P2X(3)(-/-) relative to wild-type or P2X(3)(+/-) mice. Intralumenal release of the endogenous P2X receptor ligand adenosine triphosphate did not differ between wild-type and P2X(3)(-/-) mice or change after intracolonic zymosan treatment. Responses of muscular and muscular-mucosal pelvic nerve afferents to mechanical stretch did not differ between P2X(3)(-/-) and wild-type mice. Both muscular and muscular-mucosal afferents in wild-type mice sensitized to application of an inflammatory soup, whereas only muscular-mucosal afferents did so in P2X(3)(-/-) mice. These results suggest differential roles for peripheral and central P2X(3) receptors in colon mechanosensory transduction and hypersensitivity.

Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells

Membrane-bound NTPDase2 is a member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family involved in the regulation of P2 receptor signaling. NTPDase2 has broad substrate specificity for extracellular nucleotides, but hydrolyses nucleoside 5′-triphosphates with high preference over nucleoside 5′-diphosphates. In this study, we have sought to determine how enzyme substrates acting on P2 receptors affect intracellular NTPDase2 trafficking. To achieve this, Chinese hamster ovary (CHO) cells were transiently transfected with rat-specific NTPDase2 cDNA tagged with green fluorescent protein (GFP), to allow direct visualisation of subcellular localisation and trafficking of NTPDase2. Cells were superfused with NTPDase2 substrates (ATP and UTP) and synthetic nucleotide analogues (ATPγS and ADPβS), and confocal image stacks were acquired at regular time intervals. NTPDase2 incorporation into the plasma membrane was determined by comparative analysis of fluorescence intensity in the cytosolic and membrane compartments. GFP-tagged NTPDase2 was fully functional and ATP and ATPγS induced membrane incorporation of GFP-NTPDase2 from putative intracellular stores, whilst UTP and ADPβS were ineffective. The increased ATP hydrolysis rate correlated with increased NTPDase2 trafficking to the plasma membrane. ATP-induced NTPDase2 trafficking was mediated by activation of endogenous P2X receptors involving Ca2+ entry rather than by P2Y receptor-induced release of Ca2+ from intracellular stores. Our results suggest that P2X receptor activation stimulates insertion of latent NTPDase2 into the plasma membrane. The increase in surface-located NTPDase2 may reflect a regulatory mechanism counteracting excessive stimulation and desensitisation of P2 receptors.

Characterization of ATP-gated P2X 7 receptors in fish provides new insights into the mechanism of release of the leaderless cytokine interleukin-1β

Mammalian interleukin-1β (IL-1β) is produced as a biologically inactive precursor molecule, which is proteolytically cleaved to an active form by IL-1β-converting enzyme (ICE) after the activation of P2X 7 receptor by extracellular ATP. The mechanism of IL-1β release in non-mammalian vertebrates is largely unknown, although most of the IL-1β gene sequences lack a conserved ICE recognition site. Here we have cloned the P2X 7 receptor from the bony fish seabream and compared agonist and antagonist profiles at this and other non-mammalian P2X 7 receptors expressed in HEK cells, as well in seabream SAF-1 cells expressing endogenous P2X 7 receptors. We used this information to further investigate the mechanisms of IL-1β release induced by mammalian and fish P2X 7 receptors. Despite phosphatidylserine externalization and cell permeabilization in seabream leukocytes after the addition of high BzATP concentrations, IL-1β remained unprocessed within the cell. However, activation of rat P2X 7 receptors ectopically expressed in HEK293 together with human ICE led to the specific secretion of unprocessed seabream IL-1β. In contrast, neither seabream nor zebrafish P2X 7 receptors induced the secretion of mammalian or fish IL-1β when expressed in HEK293, while a chimeric receptor harboring the ATP-binding domain of seabream P2X 7 and the intracellular region of its rat counterpart did so. These findings indicate that P2X 7 receptor-mediated activation of ICE and release of IL-1β result from different downstream signaling pathways and suggest that although the mechanisms involved in IL-1β secretion are conserved throughout evolution, distinct inflammatory signals have been selected for the secretion of this cytokine in different vertebrates.

ATP stimulates GRK-3 phosphorylation and beta-arrestin-2-dependent internalization of P2X7 receptor.

The objective of this study was to understand the mechanisms involved in P2X(7) receptor activation. Treatments with ATP or with the P2X(7) receptor-specific ligand 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) induced pore formation, but the effect was slower in CaSki cells expressing endogenous P2X(7) receptor than in human embryonic kidney (HEK)-293 cells expressing exogenous P2X(7) receptor (HEK-293-hP2X(7)-R). In both types of cells Western blots revealed expression of three forms of the receptor: the functional 85-kDa form present mainly in the membrane and 65- and 18-kDa forms expressed in both the plasma membrane and the cytosol. Treatments with ATP transiently decreased the 85-kDa form and increased the 18-kDa form in the membrane, suggesting internalization, degradation, and recycling of the receptor. In CaSki cells ATP stimulated phosphorylation of the 85-kDa form on tyrosine and serine residues. Phosphorylation on threonine residues increased with added ATP, and it increased ATP requirements for phosphorylation on tyrosine and serine residues, suggesting a dominant-negative effect. In both CaSki and in HEK-293-hP2X(7)-R cells ATP also increased binding of the 85-kDa form to G protein-coupled receptor kinase (GRK)-3, beta-arrestin-2, and dynamin, and it stimulated beta-arrestin-2 redistribution into submembranous regions of the cell. These results suggest a novel mechanism for P2X(7) receptor action, whereby activation involves a GRK-3-, beta-arrestin-2-, and dynamin-dependent internalization of the receptor into clathrin domains, followed in part by receptor degradation as well as receptor recycling into the plasma membrane.

ATP-induced [Ca(2+)](i) changes and depolarization in GH3 cells.

Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca(2+) signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line. BzATP- and ATP-induced [Ca(2+)](i) responses had EC(50) values of 18 and 651 microM, respectively. The maximal response to ATP was only 59+/-8% of that for BzATP. The BzATP-induced [Ca(2+)](i) increase was dependent upon the extracellular Ca(2+) concentration. Preincubation with oxidized ATP (oATP) nearly abolished the ATP- and BzATP-induced [Ca(2+)](i) increases. Both BzATP and ATP induced depolarization in GH3 cells, with EC(50) values of 31 microM and 1 mM, respectively. The maximal depolarization to BzATP and ATP were 152+/-21 and 146+/-16% of that elicited by 30 mM KCl. The rank order of agonist potency for [Ca(2+)](i) and depolarization responses was BzATP > > ATP >2-MeSATP and purine derivatives such as ADP, AMP, adenosine were ineffective. Neither UTP nor alpha, beta-methylene ATP showed any effect. In low-divalent conditions BzATP evoked non-desensitizing inward currents, which were reversed at approximately 0 mV. This nonselective cationic conductance was increased by repeated applications of BzATP and the cells became very permeable to NMDG. Longer applications (30 min) of BzATP stimulated ethidium bromide influx in low divalent conditions, suggesting increased permeability to larger molecules. We also identified the existence of P2X(7) mRNA on GH3 cells by using reverse transcriptase (RT)-polymerase chain reaction (PCR). These results suggest that the GH3 cells have an endogenous P2X(7) receptor and purinergic stimulation may play a potential role in neuroendocrine modulation on these cells.

Localisation of P2X receptors in human salivary gland epithelial cells and human embryonic kidney cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting and immunofluorescence.

Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human salivary gland and human embryonic kidney cell line human embryonic kidney (HEK)293 were examined for the purpose of establishing whether they expressed endogenous P2X ionotropic receptors at any stage in their cycles. HSG cells were found to express P2X1-6 subtypes using both Western blotting and immunofluorescence labeling. HEK293 cells had no detectable levels of P2X1-3 and P2X6 under normal circumstances along with very low levels of P2X4 and P2X5 but when the cells were grown past confluence then all subtypes were expressed on the surface membrane with the exception of P2X2. The results are discussed in terms of the likely influence of ATP acting as an intercellular signaling molecule.

P2X 2 characteristics of the ATP receptor coupled to [Ca 2+] i increases in cultured Purkinje neurons from neonatal rat cerebellum

P2X receptors present in cerebellar Purkinje cells have been studied by recording ATP-elicited [Ca 2+] i signals from immuno-identified (calbindin +) cells in culture using fura-2 microfluorescence. The [Ca 2+] i increases evoked by ATP were mimicked by 2MeSATP but not by α,β-meATP and other purinoceptor agonists. The selective P2X 1 antagonist diinosine pentaphosphate failed to inhibit ATP-elicited [Ca 2+] i transients, but suramin and PPADS rapidly and reversibly blocked the [Ca 2+] i responses to ATP and 2MeSATP. The IC 50 values for suramin and PPADS inhibition were 48.7±4.4 and 5.9±0.3 μM, respectively. Both antagonists blocked completely the signal elicited by ATP, revealing that there was not a separate antagonist-insensitive P2X receptor population in Purkinje cells. The effect of ATP was potentiated by Zn 2+ and H + ions. A one unit acidification from pH 7.4 to 6.4 enhanced by 172% the [Ca 2+] i transient elicited by an intermediate concentration of ATP. Conversely, alkalinization of the medium to pH 8.4 reduced the ATP response by 88%. This combination of pharmacological and modulatory properties indicates that endogenous P2X receptors present in Purkinje neurons are formed by P2X 2 subunits, rather than the more abundantly expressed P2X 4 purinoceptor subunits.

Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells.

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.