C terminus of the P2X7 receptor: treasure hunting

P2X receptor (P2XR) is a family of the ATP-gated ion channel family and can permeabilize the plasma membrane to small cations such as potassium, sodium, and calcium, resulting in cellular depolarization. There are seven P2XR that have been described and cloned, with 45% identity in amino acid sequence. Each P2X receptors has two transmembrane domains that are separated by an extracellular loop and an intracellular N and C terminus. Unlike the other P2X receptors, the P2X7R has a larger C terminus with an extra 200 amino acid residues compared with the other receptors. The C terminus of the P2X7R has been implicated in regulating receptor function including signaling pathway activation, cellular localization, protein–protein interactions, and post-translational modification (PTM). In the present review, we discuss the role of the P2X7R C terminus in regards to receptor function, describe the specific domains and motifs found therein and compare the C terminus sequence with others proteins to discover predicted domains or sites of PTM.     

A 5' intronic splice site polymorphism leads to a null allele of the P2X7 gene in 1-2% of the Caucasian population

The P2X(7) gene is important for the innate immune response but known polymorphisms do not explain all subjects with loss of P2X(7) function. A splice site mutation (g-->t) was found at position +1 of the first intron of the P2X(7) gene in 7 of 336 Caucasians and 1 of 39 subjects of Indian ethnicity. All eight subjects were heterozygous for the uncommon 1513A-->C polymorphism of the P2X(7) gene. RT-PCR and sequencing showed the splice site mutation was on the 1513C allele in the Caucasians and on the 1513A allele in the Indian subject. The splice site mutation is an inherited polymorphism and gives rise to a P2X(7) null allele in 1-2% of the Caucasian population.     

A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages

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.     

Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter

A region 2 kb upstream of exon 1 of the P2X7 gene was sequenced using DNA from nine healthy individuals who exhibited three different ATP response phenotypes (i.e. high, low and interferon gamma-inducible). Five single nucleotide polymorphisms were identified within the nine donor promoter sequences but none were associated with a specific ATP response phenotype. A P2X7 loss of function polymorphism (1513 in exon 13) was also screened for within donor DNA but no response associations were identified. ATP response phenotype was positively associated with P2X(7) receptor expression, as assessed by flow cytometry, but not with any identified receptor or promoter gene polymorphisms.     

P2X7, a critical regulator and potential target for bone and joint diseases

Abundant evidence indicted that P2X7 receptor show a essential role in human health and some human diseases including hypertension, atherosclerosis, pulmonary inflammation, tuberculosis infection, psychiatric disorders, and cancer. P2X7 receptor also has an important role in some central nervous system diseases such as neurodegenerative disorders. Recently, more research suggested that P2X7 receptor also plays a crucial role in bone and joint diseases. But the effect of P2X7 receptor on skeletal and joint diseases has not been systematically reviewed. In this article, the role of P2X7 receptor in skeletal and joint diseases is elaborated. The activation of P2X7 receptor can ameliorate osteoporosis by inducing a fine balance between osteoclastic resorption and osteoblastic bone formation. The activation of P2X7 receptor can relieve the stress fracture injury by increasing the response to mechanical loading and inducing osteogenesis. But the activation of P2X7 receptor mediates the cell growth and cell proliferation in bone cancer. In addition, the activation of P2X7 receptor can aggravate the process of some joint diseases such as osteoarthritis, rheumatoid arthritis, and acute gouty arthritis. The inhibition of P2X7 receptor can alleviate the pathological process of joint disease to some extent. In conclusion, P2X7 receptor may be a critical regulator and therapeutic target for bone and joint diseases.     

P2X7 purinoceptor alterations in dystrophic mdx mouse muscles: relationship to pathology and potential target for treatment

Duchenne muscular dystrophy (DMD) is a lethal inherited muscle disorder. Pathological characteristics of DMD skeletal muscles include, among others, abnormal Ca(2+) homeostasis and cell signalling. Here, in the mdx mouse model of DMD, we demonstrate significant P2X7 receptor abnormalities in isolated primary muscle cells and cell lines and in dystrophic muscles in vivo. P2X7 mRNA expression in dystrophic muscles was significantly up-regulated but without alterations of specific splice variant patterns. P2X7 protein was also up-regulated and this was associated with altered function of P2X7 receptors producing increased responsiveness of cytoplasmic Ca(2+) and extracellular signal-regulated kinase (ERK) phosphorylation to purinergic stimulation and altered sensitivity to NAD. Ca(2+) influx and ERK signalling were stimulated by ATP and BzATP, inhibited by specific P2X7 antagonists and insensitive to ivermectin, confirming P2X7 receptor involvement. Despite the presence of pannexin-1, prolonged P2X7 activation did not trigger cell permeabilization to propidium iodide or Lucifer yellow. In dystrophic mice, in vivo treatment with the P2X7 antagonist Coomassie Brilliant Blue reduced the number of degeneration-regeneration cycles in mdx skeletal muscles. Altered P2X7 expression and function is thus an important feature in dystrophic mdx muscle and treatments aiming to inhibit P2X7 receptor might slow the progression of this disease.     

Detection of human P2X7 nucleotide receptor polymorphisms by a novel monocyte pore assay predictive of alterations in lipopolysaccharide-induced cytokine production

The nucleotide receptor P2X(7) is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X(7) gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X(7) pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 +/- 4; AC, 42 +/- 4; and CC, 6 +/- 1-fold stimulation). Thirty of 200 healthy individuals were identified as having low P2X(7) pore activity. Seven low pore subjects were 1513 CC, 3 and 11 participants had the other known variants 946 GA and 1729 TA respectively; the remaining 9 volunteers likely have novel polymorphisms. Because platelets are a large source of extracellular ATP during inflammation, whole blood was treated ex vivo with Salmonella typhimurium LPS in the absence of exogenous nucleotides. LPS-stimulated whole blood from individuals in the low pore activity group generated reduced plasma levels of TNF-alpha (p = 0.036) and higher amounts of IL-10 (p < 0.001) relative to the high pore controls. This reduction in the TNF-alpha to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X(7) polymorphisms to regulate the LPS-induced TNF-alpha to IL-10 ratio suggests that 15% of healthy adults may exhibit anti-inflammatory mediator responses during major infectious perturbations of the immune system, which can be predicted by P2X(7) pore activity.     

Genetically dissecting <Emphasis Type="Italic">P2rx7</Emphasis> expression within the central nervous system using conditional humanized mice

The purinergic P2X7 receptor (P2X7R) has attracted considerable interest as a potential target for various central nervous system (CNS) pathologies including affective and neurodegenerative disorders. To date, the distribution and cellular localization of the P2X7R in the brain are not fully resolved and a matter of debate mainly due to the limitations of existing tools. However, this knowledge should be a prerequisite for understanding the contribution of the P2X7R to brain disease. Here, we generated a genetic mouse model by humanizing the P2X7R in the mouse as mammalian model organism. We demonstrated its functionality and revealed species-specific characteristics of the humanized receptor, compared to the murine ortholog, regarding its receptivity to activation and modulation by 2′,3′-O-(benzoyl-4-benzoyl)-adenosine 5′-triphosphate (BzATP) and trifluoperazine (TFP). This humanized P2rx7 allele is accessible to spatially and temporally controlled Cre recombinase-mediated inactivation. In contrast to previously generated knockout (KO) mice, none of the described P2rx7 splice variants evade this null allele. By selective disruption and assessment of human P2RX7 expression in different brain regions and cell types, we were able to demonstrate that the P2X7R is specifically expressed in glutamatergic pyramidal neurons of the hippocampus. Also, P2X7R is expressed in major non-neuronal lineages throughout the brain, i.e., astrocytes, oligodendrocytes, and microglia. In conclusion, this humanized mouse model provides the means for detailed assessment of human P2X7R function in vivo including evaluation of agonists or antagonists. In addition, this conditional allele will enable future loss-of-function studies in conjunction with mouse models for CNS disorders.     

The Pro-451 to Leu polymorphism within the C-terminal tail of P2X7 receptor impairs cell death but not phospholipase D activation in murine thymocytes

The P2X family of ATP receptors (P2XR) are ligandgated channels that have been proposed to regulate cell death of immature thymocytes. However, the nature of the P2XR subtype involved has been controversial until recently. In agreement with previous studies, we found that extracellular ATP (ATPe) induces a caspase-dependent apoptosis of BALB/c thymocytes, as observed by DNA fragmentation. Additionally, ATPe induces a predominant caspase-independent thymocytes lysis characterized by plasma membrane disruption. Both responses to ATPe can be induced by a potent P2X7R agonist, benzoylbenzoyl-ATP, whereas P2X7R antagonists, oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, inhibited the effect of ATPe. These results are further supported by observations where disruption of the P2X7R gene (P2X7R(-/-) mice) completely abolishes thymocytes death induced by ATPe. Interestingly, the natural P451L mutation in the C-terminal tail of P2X7R present in C57BL/6 mice, which impairs ATPe-dependent pore formation in T lymphocytes, significantly reduces thymocytes death triggered by ATPe. Furthermore, we found that P2X7R from BW5147 thymoma cells also harbors this point mutation, accounting for their insensitivity to ATPe-induced cell death. Concentrations of ATPe effective in inducing cell death also increase phosphatidylcholine-hydrolyzing phospholipase D (PC-PLD) activity in BALB/c thymocytes through the stimulation of P2X7R. However, in contrast to ATPe-induced cell death, PC-PLD activation is totally Ca(2+)-dependent. Moreover, the stimulation of PC-PLD by ATPe is not affected by the P451L mutation present in C57BL/6 thymocytes and BW5147 cells, suggesting that cell death and PC-PLD activity are regulated through distinct domains of the P2X7R. Finally, the inhibition of ATPe-induced PC-PLD stimulation does not affect thymocytes death. Altogether, these data suggest that P2X7R-induced thymocytes death is independent of the stimulation of PC-PLD activity.     

Protein kinase C regulation of P2X3 receptors is unlikely to involve direct receptor phosphorylation

P2X receptors (P2XR) act as ligand-gated, cation-selective ion channels. A common characteristic of all seven P2X family members is a conserved consensus sequence for protein kinase C (PKC)-mediated phosphorylation in the intracellular N-terminus of the receptor. Activation of PKC has been shown to enhance currents through P2X(3)R, however the molecular mechanism of this potentiation has not been elucidated. In the present study we show that activation of PKC can enhance adenosine triphosphate (ATP)-mediated Ca(2+) signals approximately 2.5-fold in a DT-40 3KO cell culture system (P2 receptor null) transiently overexpressing P2X(3)R. ATP-activated cation currents were also directly studied using whole cell patch clamp techniques in HEK-293 cells, a null background for ionotropic P2XR. PKC activation resulted in a approximately 8.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X(3)R cDNA, but had no effect on currents through either P2X(4)R- or P2X(7)R-transfected cells. P2X(3)R-transfected HEK-293 cells were metabolically labeled with (32)PO(4)(-) and following treatment with phorbol-12-myristate-13-acetate (PMA) and subsequent immunoprecipitation, there was no incorporation of (32)PO(4)(-) in bands corresponding to P2X(3)R. Similarly, in vitro phosphorylation experiments, utilizing purified PKC catalytic subunits failed to establish phosphorylation of either P2X(3)R or P2X(3)R-EGFP. These data indicate that PKC activation can enhance both the Ca(2+) signal as well as the cation current through P2X(3)R, however it appears that the regulation is unlikely to be a result of direct phosphorylation of the receptor.     

Genetics of the P2X7 receptor and human disease

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.     

Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca²⁺ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.     

Mouse dendritic cells express the P2X7 purinergic receptor: characterization and possible participation in antigen presentation.

Immune cells express P2 purinoceptors of the P2Y and P2X subtypes. In the present work, we show that three dendritic cell (DC) lines, D2SC/1, CB1, and FSDC, representative of immature DCs, express the P2X7 (formerly P2Z) receptor, as judged from RT-PCR amplification, reactivity to a specific antiserum, and pharmacological and functional evidence. Receptor expression is higher in FSDC cells, a cell line that is functionally more mature than D2SC/1 and CB1. From the wild-type DC population, we selected cell clones lacking the P2X7R (P2X7less). We also used a P2XR blocker, oxidized ATP, to irreversibly inhibit the P2X7R. Ability of P2X7less FSDCs or of oxidized ATP-inhibited FSDCs to stimulate Ag-specific TH lymphocytes was severely decreased although Ag endocytosis was minimally affected. During coculture with TH lymphocytes, wild-type FSDC secreted large amounts of IL-1beta. Release of this cytokine was reduced in P2X7less DCs. These data show that DCs express the P2X7 purinoceptor and suggest a correlation between P2X7R expression and Ag-presenting activity.     

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.     

白血病细胞系来源的P2X7受体的功能研究

P2X7受体是ATP门控的离子通道。从白血病细胞系J6-1细胞中扩增P2X7受体编码区全序列,克隆到pTARGET真核表达载体,经DNA序列分析后转染Ramos细胞,获得稳定表达细胞株;应用RT-PCR、Westernblot和流式细胞术检测P2X7受体在Ramos中的表达;荧光分光光度计检测P2X7受体介导的胞内钙离子浓度变化。结果显示,J6-1细胞来源的P2X7受体在第559位有一个A→G的有义突变,导致Asn187→Asp187,可在Ramos细胞中表达,在特异性激动剂BzATP作用下可引起胞内钙离子浓度的升高,但所需激动剂浓度高于常规浓度。     

Epithelial membrane proteins induce membrane blebbing and interact with the P2X7 receptor C terminus

The binding of extracellular ATP to the P2X(7) receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1beta secretion and eventual death. The latter three effects are unique to the P2X(7) receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X(7) receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X(7) receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X(7) was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathione S-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X(7). The P2X(7) receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X(7) receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X(7) C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X(7) receptor activation.     

Involvement of chloride in apoptotic cell death induced by activation of ATP-sensitive P2X7 purinoceptor

The ATP-gated P2X(7) receptor is a plasma membrane receptor belonging to the family of P2X purinoceptors. Its activation leads to multiple downstream events including influx of ions, pore formation to allow the passage of larger molecular weight species, and cell death by apoptosis and/or necrosis. The cell death is thought to be correlated with the pore formation but does not directly result from the dilatation of pores. We have generated and characterized a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor. In this study, we investigated the mechanism of P2X(7) receptor-induced cell death using this clone. Treatment with P2X(7) receptor agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP induced depolarization of membrane potential, pore formation, and cell shrinkage, an early hallmark of apoptosis in the buffer containing physiological concentrations of ions. Analysis by flow cytometry revealed that the activity of pore formation in shrunk cells was much higher than in non-shrunk cells. The activation of P2X(7) receptor also caused the release of lactate dehydrogenase from cells. The P2X(7) receptor-mediated cell shrinkage and lactate dehydrogenase release were blocked when media Cl(-) was replaced with gluconate. However, removal of extracellular Cl(-) did not affect plasma membrane depolarization and pore formation by treatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP. Therefore we concluded that pore formation plays a critical role in the P2X(7) receptor-induced apoptotic cell death and that this is mediated by extracellular Cl(-) influx.     

An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor

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 receptor have been observed, explained in part by (7)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).     

P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region

The importance of the cytosolic C-terminal region of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake, whereas those expressing the P2X7R truncated at position 582 give wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region between residues 551 and 581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues within this region could also abolish receptor function and cell surface expression, suggesting that this region may participate in regulating the surface expression of the pore-forming P2X7R.