Exacerbation of experimental autoimmune encephalomyelitis in P2X7R-/- mice: evidence for loss of apoptotic activity in lymphocytes

The purinergic receptor P2X7R is a nucleotide-gated ion channel that has been proposed to function as a major regulator of inflammation. In this study we examined the role of this receptor in regulating inflammation in the CNS by determining the effects of the loss of this receptor (P2X7R-/-) on experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We show here that P2X7R-/- mice developed more severe clinical and pathological expression of EAE than wild type (WT) controls and that spleen and lymph node cells from P2X7R-/- mice proliferated more vigorously to Ag in vitro. Bone marrow (BM) radiation chimeras revealed that enhanced susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R-/- BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. Coculture of P2X7R-/- macrophages with WT lymphocytes and vice versa showed that enhanced proliferative activity resided within the P2X7R-/- lymphocyte population and correlated with reduced levels of IFN-gamma and NO and apoptosis of lymphocytes. mRNA and protein for IFN-gamma were also significantly reduced in the CNS of P2X7R-/- mice with EAE. FACS analysis of cells isolated from the CNS showed significantly fewer annexin V/propidium iodide-positive lymphocytes in the CNS of P2X7R-/- mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. From these data we conclude that enhanced susceptibility of P2X7R-/- mice to EAE reflects a loss of apoptotic activity in lymphocytes, supporting an important role for this receptor in lymphocyte homeostasis.     

POM-1 inhibits P2 receptors and exhibits anti-inflammatory effects in macrophages

Extracellular nucleotides can modulate the immunological response by activating purinergic receptors (P2Rs) on the cell surface of macrophages, dendritic, and other immune cells. In particular, the activation of P2X7R can induce release of cytokines and cell death as well as the uptake of large molecules through the cell membrane by a mechanism still poorly understood. Polyoxotungstate-1 (POM-1) has been proposed as a potent inhibitor of ecto-nucleotidases, enzymes that hydrolyze extracellular nucleotides, regulating the activity of P2Rs. However, the potential impact of POM-1 on P2Rs has not been evaluated. Here, we used fluorescent dye uptake, cytoplasmic free Ca²⁺ concentration measurement, patch-clamp recordings, scanning electron microscopy, and quantification of inflammatory mediators to investigate the effects of POM-1 on P2Rs of murine macrophages. We observed that POM-1 blocks the P2YR-dependent cytoplasmic Ca²⁺ increase and has partial effects on the cytoplasmic Ca²⁺, increasing dependence on P2XRs. POM-1 can inhibit the events related with ATP-dependent inflammasome activation, anionic dye uptake, and also the opening of large conductance channels, which are associated with P2X7R-dependent pannexin-1 activation. On the other hand, this compound has no effects on cationic fluorescent dye uptake, apoptosis, and bleb formation, also dependent on P2X7R. Moreover, POM-1 can be considered an anti-inflammatory compound, because it prevents TNF-α and nitric oxide release from LPS-treated macrophages.     

The Hyposensitive N187D P2X7 Mutant Promotes Malignant Progression in Nude Mice

Nucleotides are new players in the intercellular communication network. P2X7 is a member of the P2X family of receptors, which are ATP-gated plasma membrane ion channels with diverse biological functions. Abnormal expression and dysfunction of P2X7 have been reported in leukemias. Here, we report a new P2X7 mutant (an A⁵⁵⁹-to-G substitution causing N187D P2X7) cloned from J6-1 leukemia cells. The characteristics of N187D P2X7 were studied by establishing stably transfected K562 cell lines. Our results show that N187D P2X7 required a higher concentration of agonist for its activation, leading to Ca²⁺ influx (EC₅₀ = 293.3 ± 6.6 μm for the mutant and 93.6 ± 2.2 μm for wild-type P2X7) and ERK phosphorylation, which were not caused by differential cell-surface expression or related to high ATPase activity on the cell surface and in the extracellular space. K562 cells expressing this N187D mutant showed a proliferative advantage and reduced pro-apoptosis effects in vitro and in vivo. Furthermore, elevated angiogenesis and CD206-positive macrophage infiltration were found in tumor tissues formed by K562-M cells. In addition, higher expression of VEGF and MCP1 could be detected in tumor tissues formed by K562-M cells. Our results suggest that N187D P2X7, representing mutants hyposensitive to agonist, might be a positive regulator in the progression of hematopoietic malignancies.     

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.     

Contributions of the C-terminal domain to the control of P2X receptor desensitization

The P2X purinergic receptor channels (P2XRs) differ among themselves with respect to the rates of desensitization during prolonged agonist stimulation. Here we studied the desensitization of recombinant channels by monitoring the changes in intracellular free Ca(2+) concentration in cells stimulated with ATP, the native and common agonist for all P2XRs. The focus in our investigations was on the relevance of the P2XR C terminus in controlling receptor desensitization. When expressed in GT1 cells, the P2XRs desensitized with rates characteristic to each receptor subtype: P2X(1)R = P2X(3)R > P2X(2b)R > P2X(4)R > P2X(2a)R > P2X(7)R. A slow desensitizing pattern of P2X(2a)R was mimicked partially by P2X(3)R and fully by P2X(4)R when the six-amino acid sequences of these channels located in the cytoplasmic C terminus were substituted with the corresponding arginine 371 to proline 376 sequence of P2X(2a)R. Changing the total net charge in the six amino acids of P2X(4)R to a more positive direction also slowed the receptor desensitization. On the other hand, substitution of arginine 371-proline 376 sequence of P2X(2a)R with the corresponding sequences of P2X(1)R, P2X(3)R, and P2X(4)R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X(2a)R and mutant P2X(3)R having the C-terminal six amino acids of P2X(2a)R at its analogous position resulted in a functional channel whose desensitization was significantly delayed. These results suggest that composition of the C-terminal six-amino acid sequence and its electrostatic force influence the rate of receptor desensitization.     

P2X7 receptor signaling promotes inflammation in renal parenchymal cells suffering from ischemia-reperfusion injury

Extracellular adenosine triphosphate (ATP) and its receptor, P2X7 receptor (P2X7R), are playing an important role in the pathological process of renal ischemia-reperfusion injury, but their underlying mechanism remains unclear. Also, the effects of tubular epithelium-expressed P2X7 receptor on ischemia acute kidney injury is still unknown. The aim of this study is to clarify if this mechanism involves the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the renal tubular epithelial cells. In our research, we used male C57BL/6 wild type and P2X7R (−/−) mice, cultured human proximal tubular epithelial cells, and kidneys from acute kidney injury patients. Mice underwent for unilateral nephrectomy combined with the lateral renal pedicle clamping. Cultured cells were subjected to hypoxia/reoxygenation or ATP. Apyrase and A438079 were used to block the extracellular ATP/P2X7 receptor pathway. We also constructed radiation-induced bone marrow (BM) chimeras by using P2X7R (−/−) mice and P2X7R (+/+) wild-type mice. P2X7 receptor deficiency protected from renal ischemia-reperfusion injury and attenuated the formation of NLRP3 inflammasome. By using BM chimeras, we found a partial reduction of serum creatinine and less histological impairment in group wild-type BM to P2X7R (−/−) recipient, compared with group wild-type BM to wild-type recipient. In renal tubular epithelial cells, hypoxia/reoxygenation induced ATP release and extracellular ATP depletion reduced the expression of active IL-1β. ATP activated the NLRP3 inflammasome in renal tubular epithelial cells, which were blunted by transient silence of P2X7 receptor, as well as by P2X7 receptor blocking with A438079. In human samples, we found that patients with Stage 3 AKI had higher levels of P2X7 receptor expression than patients with Stage 1 or Stage 2. Extracellular ATP/P2X7 receptor axis blocking may protect renal tubular epithelial cells from ischemia-reperfusion injury through the regulation of NLRP3 inflammasome.Subject terms: Membrane proteins, Mechanisms of disease, Acute kidney injury     

Abnormal expression of P2X family receptors in Chinese pediatric acute leukemias

Nucleotides are new players in intercellular communication network. P2X family receptors are ATP-gated plasma membrane ion channels with diverse biological functions. Their distribution patterns and significance in pediatric leukemias have not been established. Here we investigated the expression of P2X receptors in BMMC samples from Chinese pediatric acute leukemias. Real-time PCR and Western blot results showed that P2X1, P2X4, P2X5 and P2X7 receptors were simultaneously over expressed in leukemias compared with controls, whereas P2X2, P2X3 and P2X6 were absent or marginally expressed in both groups. It was worth noting that the co-expression feature of them, especially between P2X4 and P2X7, could be observed and the highest expression of P2X7 was detected in relapsed patients. Moreover, concomitant decrease of P2X4, P2X5 and P2X7 expressions was observed at CR stage in a follow-up study. Functional P2X7 was also verified. These results suggested that P2X1, P2X4, P2X5 and P2X7 were hematopoiesis-related P2X receptors, and their signaling, especially for P2X7, might play important roles in pediatric leukemias. P2X receptors might co-operatively contribute to the malignant phenotype in human pediatric leukemias.     

Functional relevance of aromatic residues in the first transmembrane domain of P2X receptors

The functional relevance of aromatic residues in the upper part of the transmembrane domain-1 of purinergic P2X receptors (P2XRs) was examined. Replacement of the conserved Tyr residue with Ala had a receptor-specific effect: the P2X1R was non-functional, the P2X2R, P2X4R, and P2X3R exhibited enhanced sensitivity to ATP and αβ-meATP accompanied by prolonged decay of current after washout of agonists, and the P2X7R sensitivity for agonists was not affected, though decay of current was delayed. The replacement of the P2X4R-Tyr42 with other amino acids revealed the relevance of an aromatic residue at this position. Mutation of the neighboring Phe and ipsilateral Tyr/Trp residues, but not the contralateral Phe residue, also affected the P2X2R, P2X3R, and P2X4R function. Double mutation of ipsilateral Tyr42 and Trp46 P2X4R residues restored receptor function, whereas the corresponding P2X2R double mutant was not functional. In contrast, mutation of the contralateral Phe48 residue in the P2X4R-Y42A mutant had no effect. These results indicate that aromatic residues in the upper part of TM1 play important roles in the three-dimensional structure of the P2XRs and that they are required not only for ion conductivity but also for specificity of agonist binding and/or channel gating.     

Homotrimeric complexes are the dominant assembly state of native P2X7 subunits

P2X receptors (P2XRs) are trimeric ATP-gated cation channels. Seven subunits have been cloned. P2X4 and P2X7 subunits show overlapping expression and both subunits are involved in pathophysiological processes such as inflammatory and neuropathic pain. A recent study provides evidence for heteromeric P2X4/7Rs.In this study, subtype-specific antibodies in combination with BN-PAGE are used to directly visualize P2XR complexes solubilized from membrane extracts of native tissues. The results show specific P2X7R and P2X4R staining in many tissues. The P2X7 complex has a clearly different size than the P2X4 complex but is likewise composed of three subunits. No complexes corresponding to more than three subunits could be detected. Also, no complexes of intermediate size or reactive to both antibodies were detected. These data suggest that either heteromerization between P2X4 and P2X7 subunits results not in stable heteromeric complexes or P2X4/7 heteromers do not represent a dominant subtype in the tissues investigated.     

A consensus segment in the M2 domain of the hP2X7 receptor shows ion channel activity in planar lipid bilayers and in biological membranes

The P2X₇ receptor (P2X₇R) is an ATP-gated, cation-selective channel permeable to Na⁺, K⁺ and Ca²⁺. This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X₇R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X₇R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X₇R. The conserved part of the M2 domain of P2X₇R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.     

Altered cytokine production in mice lacking P2X(7) receptors

The P2X(7) receptor (P2X(7)R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1 beta post-translational processing, we have generated a P2X(7)R-deficient mouse line. P2X(7)R(-/-) macrophages respond to lipopolysaccharide and produce levels of cyclooxygenase-2 and pro-IL-1 beta comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1 beta produced by the P2X(7)R(-/-) cells is not externalized or activated by caspase-1. Nigericin, an alternate secretion stimulus, promotes release of 17-kDa IL-1 beta from P2X(7)R(-/-) macrophages. In response to in vivo lipopolysaccharide injection, both wild-type and P2X(7)R(-/-) animals display increases in peritoneal lavage IL-6 levels but no detectable IL-1. Subsequent ATP injection to wild-type animals promotes an increase in IL-1, which in turn leads to additional IL-6 production; similar increases did not occur in ATP-treated, LPS-primed P2X(7)R(-/-) animals. Absence of the P2X(7)R thus leads to an inability of peritoneal macrophages to release IL-1 in response to ATP. As a result of the IL-1 deficiency, in vivo cytokine signaling cascades are impaired in P2X(7)R-deficient animals. Together these results demonstrate that P2X(7)R activation can provide a signal that leads to maturation and release of IL-1 beta and initiation of a cytokine cascade.     

P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8⁺ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8⁺ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8⁺ T cell immunity.     

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca²⁺, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca²⁺ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca²⁺ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X₃R > P2X_2b + X₄R > P2X_2bR > P2X_2a + X₄R > P2X₄R > P2X_2aR > P2X₇R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca²⁺ influx because of the coactivation of endogenously expressed Ca²⁺-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca²⁺ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca²⁺ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca²⁺ signals were not affected by voltage-gated Ca²⁺ influx and removal of extracellular sodium. Ca²⁺ signals reflected well the receptor-specific EC₅₀ values for ATP and the extracellular Zn²⁺ and pH sensitivities of P2XRs. These results indicate that intracellular Ca²⁺ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals     

A monoclonal antibody against an antigen present on mouse macrophages and absent from monocytes

Summary A monoclonal antibody (BM8) raised in the rat against cultured mouse bone marrow-derived macrophages reacted only with macrophages and not with granulocytes, mast cells, platelets, lymphocytes, fibroblasts, and endothelial cells. BM8 did not detect blood monocytes. In cultured bone-marrow cells, expression of BM8-antigen was found on a few macrophages after one day of culture and reached its maximum level with 80% positive macrophages after 7–10 days of culture. The antibody BM8 belonged to the IgG_2a subclass, was non-cytotoxic and directed against a 125 kD membrane antigen. In cryostat sections of normal mouse tissues (spleen, lymph node, thymus, liver, skin) BM8 detected tissue-fixed macrophages and Langerhans cells in the skin. In spleen and lymph node, BM8 reacted with macrophages in the red pulp and in the medullary cords, respectively, but not with heavily phagocytosing marginal-zone macrophages, as revealed by in-vivo phagocytosis of colloidal carbon. In granulomata induced by complete Freund's adjuvant, BM8 detected inflammatory macrophages but not epithelioid cells. Thus, antibody BM8 detected a differentiation antigen expressed only on mature, tissue-fixed macrophages.     

A consensus segment in the M2 domain of the hP2X(7) receptor shows ion channel activity in planar lipid bilayers and in biological membranes

The P2X(7) receptor (P2X(7)R) is an ATP-gated, cation-selective channel permeable to Na(+), K(+) and Ca(2+). This channel has also been associated with the opening of a non-selective pore that allows the flow of large organic ions. However, the biophysical properties of the P2X(7)R have yet to be characterized unequivocally. We investigated a region named ADSEG, which is conserved among all subtypes of P2X receptors (P2XRs). It is located in the M2 domain of hP2X(7)R, which aligns with the H5 signature sequence of potassium channels. We investigated the channel forming ability of ADSEG in artificial planar lipid bilayers and in biological membranes using the cell-attached patch-clamp techniques. ADSEG forms channels, which exhibit a preference for cations. They are voltage independent and show long-term stability in planar lipid bilayers as well as under patch-clamping conditions. The open probability of the ADSEG was similar to that of native P2X(7)R. The conserved part of the M2 domain of P2X(7)R forms ionic channels in planar lipid bilayers and in biological membranes. Its electrophysiological characteristics are similar to those of the whole receptor. Conserved and hydrophobic part of the M2 domain forms ion channels.     

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.     

Lysophosphatidylcholine potentiates Ca2+ influx, pore formation and p44/42 MAP kinase phosphorylation mediated by P2X7 receptor activation in mouse microglial cells

The P2X7 receptor (P2X7R) is an ATP-gated ion channel highly expressed in microglia. P2X7R plays important roles in inflammatory responses in the brain. However, little is known about the mechanisms regulating its functions in microglia. Lysophosphatidylcholine (LPC), an inflammatory phospholipid that promotes microglial activation, may have some relevance to P2X7R signaling in terms of microglial function. In this study, we examined its effects on P2X7R signaling in a mouse microglial cell line (MG6) and primary microglia. LPC facilitated the sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through P2X7R channels activated by ATP or BzATP. The potentiated increase in [Ca(2+)](i) was actually inhibited by P2X7R antagonists, brilliant blue G and oxidized ATP. The potentiating effect of LPC was not observed with P2Y receptor systems, which are also expressed in MG6 cells. G2A, a receptor for LPC, was expressed in MG6 cells, but not involved in the facilitating effect of LPC on the P2X7R-mediated change in [Ca(2+)](i). Furthermore, LPC enhanced the P2X7R-associated formation of membrane pores and the activation of p44/42 mitogen-activated protein kinase. These results suggest that LPC may regulate microglial functions in the brain by enhancing the sensitivity of P2X7R.     

Contribution of P2X4 Receptors to Ethanol Intake in Male C57BL/6 Mice

P2X receptors (P2XRs) are a family of cation-permeable ligand-gated ion channels activated by synaptically released extracellular adenosine 5′-triphosphate. The P2X4 subtype is abundantly expressed in the central nervous system and is sensitive to low intoxicating ethanol concentrations. Genetic meta-analyses identified the p2rx4 gene as a candidate gene for innate alcohol intake and/or preference. The current study used mice lacking the p2rx4 gene (knockout, KO) and wildtype (WT) C57BL/6 controls to test the hypothesis that P2X4Rs contribute to ethanol intake. The early acquisition and early maintenance phases of ethanol intake were measured with three different drinking procedures. Further, we tested the effects of ivermectin (IVM), a drug previously shown to reduce ethanol’s effects on P2X4Rs and to reduce ethanol intake and preference, for its ability to differentially alter stable ethanol intake in KO and WT mice. Depending on the procedure and the concentration of the ethanol solution, ethanol intake was transiently increased in P2X4R KO versus WT mice during the acquisition of 24-h and limited access ethanol intake. IVM significantly reduced ethanol intake in P2X4R KO and WT mice, but the degree of reduction was 50 % less in the P2X4R KO mice. Western blot analysis identified significant changes in γ-aminobutyric acid_A receptor α1 subunit expression in brain regions associated with the regulation of ethanol behaviors in P2X4R KO mice. These findings add to evidence that P2X4Rs contribute to ethanol intake and indicate that there is a complex interaction between P2X4Rs, ethanol, and other neurotransmitter receptor systems.