Antagonism by the suramin analogue NF279 on human P2X(1) and P2X(7) receptors

The effect of the suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3 , 5-naphthalenetrisulfonic acid) (NF279) was analyzed on human P2X(1) and P2X(7) receptor subtypes (human P2X(1) and human P2X(7)) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. At activating ATP concentrations of 1 microM (human P2X(1)) and 10 microM ATP (human P2X(7)), IC(50) values of 0.05 microM and 2.8 microM were found for human P2X(1) and human P2X(7) receptors, respectively. An increase in the activating [ATP] shifted the NF279 concentration-inhibition curve rightwards for both receptors. NF279 slowed the activation of both human P2X(1) and human P2X(7) as well as the desensitization of human P2X(1). The data support a model in which desensitization of P2X(1) is dependent on preceding activation of these P2X receptors. It is concluded that NF279 acts as a competitive antagonist with much higher potency at human P2X(1) than at P2X(7) receptors. NF279 may hence be suited to discriminate between both receptors in native tissues.     

Profiling at recombinant homomeric and heteromeric rat P2X receptors identifies the suramin analogue NF449 as a highly potent P2X1 receptor antagonist

P2X receptors are cation channels gated by extracellular ATP and related nucleotides. Because of the widespread distribution of P2X receptors and the high subtype diversity, potent and selective antagonists are needed to dissect their roles in intact tissues. Based on suramin as a lead compound, several derivates have been described that block recombinant P2X receptors with orders of magnitude higher potency than suramin. Here we characterized the suramin analogue 4,4',4',4'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) with respect to its potency to antagonize ATP or alphabeta-methyleneadenosine 5'-trisphosphate-induced inward currents of homomeric rat P2X(1)-P2X(4) receptors or heteromeric P2X(1 + 5) and P2X(2+3) receptors, respectively. NF449 most potently blocked P2X(1) and P2X(1 + 5) receptors with IC(50) values of 0.3 nM and 0.7 nM, respectively. Three to four orders of magnitude higher NF449 concentrations were required to block homomeric P2X(3) or heteromeric P2X(2 + 3) receptors (IC(50) 1.8 and 0.3 microM, respectively). NF449 was least potent at homomeric P2X(2) receptors (IC(50) 47 microM) and homomeric P2X(4) receptors (IC(50) > 300 microM). Altogether, these results characterize NF449 as the so far most potent and selective antagonist of receptors incorporating the P2X(1) subunit such as the P2X(1) homomer and the P2X(1 + 5) heteromer.     

The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range

The role of ATP-stimulated P2X₁ receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y₁ or P2Y₁₂ receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X₁ receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X₁ antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on meATP (P2X₁ receptors)-induced or ADP (P2Y₁ receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y₁ & P2Y₁₂ receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8,8,8-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X₁ antagonist reported to date, blocking meATP-induced Ca²⁺ increases and shape change in a concentration-dependent manner, with a pA₂ of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X₁ receptor displayed the following order : NF864>NF449NF110>NF023=MK-HU1=suramin. A different antagonistic profile was observed for ADP-induced Ca²⁺ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X₁ responses, with the order NF110>NF449NF864MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X₁ receptors in thrombosis and haemostasis, NF864 being the most promising compound.     

Properties of native P2X receptors in rat trigeminal mesencephalic nucleus neurones: lack of correlation with known, heterologously expressed P2X receptors

Trigeminal mesencephalic nucleus (MNV) neurones express functional P2X receptors. In order to determine the molecular identity of the P2X receptors in this nucleus we have used whole cell patch clamp recording of P2X receptor-mediated currents to determine the pharmacological properties of the receptors, and have compared them with those of cloned P2X receptor subunits. The purine nucleotides ATP (300 microM), ATP-gamma-S (30 microM) and alphabetameATP (300 microM) evoked inward currents in all MNV neurones whereas alphabetameADP (300 microM) did not. betagammame-L-ATP (300 microM) evoked only a small ( approximately 20 pA) current in 3 out of 6 MNV neurones. The P2X receptor antagonist TNP-ATP (10 nM-10 microM) and raised extracellular Ca(2+) (8 and 30 mM) reduced, but did not abolish, the current evoked by ATP-gamma-S. The current remaining in TNP-ATP was insensitive to blockade by raised Ca(2+). These properties suggest that MNV neurones do not express homomeric P2X(3), P2X(4) or P2X(6) receptors. Whilst the TNP-ATP-insensitive ATP-gamma-S-evoked current has many characteristics similar to both homomeric P2X(2) and P2X(5) receptors, its insensitivity to blockade by raised Ca(2+) is difficult to reconcile with the receptor being a P2X(2) or P2X(5) homomeric channel. More likely, the receptor is a heteromer that comprises either or both of these subunits. The TNP-ATP-sensitive component of the ATP-gamma-S-evoked current is dissimilar to known cloned homomeric or heteromeric P2X receptors.     

Antagonistic properties of four suramin-related compounds at vascular purine P2X receptors in the pithed rat

The dose-response curve for the vasopressor effect of alpha, beta-methylene ATP in pithed rats was influenced by four suramin-related drugs (each at 100 mumol/kg). The curve was shifted to the right by a factor of 8 by 'compound 3' and by a factor of 2 by two other derivatives, but was not affected by the fourth analogue. Compound 3 had no effect on the vasopressor response to noradrenaline or neuropeptide Y. In conclusion, compound 3 is a purine P2X receptor antagonist which is approximately as potent as suramin, and which, like suramin, does not exhibit antagonistic properties at alpha-adrenoceptors and neuropeptide y receptors.     

The suramin analogue NF279 is a novel and potent antagonist selective for the P2X(1) receptor

The suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)) bis(1,3,5-naphthalenetrisul fonic acid) (NF279) was analysed with respect to its potency and P2X receptor subtype selectivity. Two-electrode voltage-clamp measurements were performed with Xenopus laevis oocytes expressing homomultimeric rat P2X(1), P2X(2), P2X(3) and human P2X(4) receptors. For the fast desensitising P2X(1) and P2X(3) receptors, IC(50) values strongly depended on whether oocytes were pre-incubated with NF279 prior to ATP superfusion or exposed to NF279 simultaneously with ATP. With a 10 s pre-incubation period of NF279, IC(50) values of 19 nM and 1.62 microM were obtained for rat P2X(1) and P2X(3), respectively. Without pre-incubation, IC(50) values amounted to 2 microM and 85.5 microM for P2X(1) and P2X(3), respectively. For the non-desensitising rat P2X(2) receptor NF279 appeared to act as a competitive antagonist with an IC(50) value of 0.76 microM and a K(B) value of 0.36 microM, as derived from Schild analysis. P2X(4) receptors were the least sensitive subtypes for NF279 (IC(50)>300 microM). The antagonism was fully reversible at all P2X subtypes analysed. Our results indicate that NF279 is a potent P2X(1) receptor-selective and reversible antagonist.     

NF449: a subnanomolar potency antagonist at recombinant rat P2X1 receptors

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC50<3.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 > P2X3 > P2Y1 > P2Y2 > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.     

Use-dependent inhibition of the skeletal muscle ryanodine receptor by the suramin analogue NF676

The skeletal muscle Ca2+ release channel, the ryanodine receptor, is activated by the trypanocidal drug suramin via the calmodulin-binding site. As calmodulin activates and inhibits the ryanodine receptor depending on whether Ca2+ is absent or present, suramin analogues were screened for inhibition of the ryanodine receptor. Up to 300 microM, the novel suramin analogue, 4,4'-(carbonyl-bis(imino-4,1-phenylene-(2,5-benzimidazolylene)carbonylimino))-bis-benzenesulfonic acid disodium salt (NF676) was not able to significantly inhibit the basal [3H]ryanodine binding. However, kinetic analysis of the high affinity [3H]ryanodine binding elucidates a time-dependent increment of inhibition by NF676, which is indicative for an open channel blocker. Moreover, the ryanodine receptor was much more sensitive towards inhibition by NF676 when preactivated with caffeine or the nonhydrolysable ATP analogue, adenylyl-imidodiphosphate. Nonetheless, the suramin activated ryanodine receptor was not susceptible towards high-affinity NF676 inhibition, indicating an allosteric hindrance between the binding sites of suramin and NF676. In the line of this finding, NF676 per se was not capable to elute the purified ryanodine receptor from a calmodulin-Sepharose, but it prevented the elution by suramin. Other than suramin, NF676 did not inhibit the Ca2+ ATPase of the sarcoplasmic reticulum. However, suramin-induced Ca2+ release from sarcoplasmic reticulum was completely abrogated by preincubation with NF676. Taken together, we conclude from these data that NF676 represents a novel lead compound as a potent use-dependent blocker of the skeletal muscle ryanodine receptor via an allosteric interaction with the suramin-binding site.     

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

BACKGROUND AND PURPOSE

The P2X receptor family consists of seven subunit types – P2X1–P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits – P2X2 and P2X4, and P2X4 and P2X7.

EXPERIMENTAL APPROACH

We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging.

KEY RESULTS

Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation.

CONCLUSIONS AND IMPLICATIONS

We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.     

Expression level dependent changes in the properties of P2X2 receptors

The currents of P2X(2) receptors expressed in Xenopus oocytes or HEK293 cells show significant cell-to-cell variation in many properties including the rate of desensitization and the magnitude of potentiation by zinc or acidic pH. In this study, we examined whether differences in expression levels underlie this variability. We injected Xenopus oocytes with different concentrations of RNA encoding rat P2X(2) to give a wide range of maximum current amplitudes, and then measured the potentiation of responses to 10 micro M adenosine 5'-triphosphate (ATP) by zinc or acidic pH.Individual oocytes showed potentiation ratios that ranged from 1.4- to 25-fold. Oocytes with small amplitude responses to a saturating concentration of ATP tended to have larger potentiation ratios than oocytes with large amplitude responses. This phenomenon was explained by an inverse correlation between the EC(50) for ATP and the maximum current amplitude, with the EC(50) decreasing from about 37 to 7 micro M as expression level increased. In contrast, the Hill coefficient was not correlated with the maximum current amplitude. Truncated receptors lacking the last 76 amino acids also showed an inverse correlation between the EC(50) and the maximum current amplitude. Thus, the interactions that cause expression-dependent changes in P2X(2) receptor properties must involve domains proximal to position H397.     

Neuronal P2X receptors: localisation and functional properties

ATP is a co-transmitter in the central and peripheral nervous system. Extracellular ATP exerts its effects via ionotropic (P2X), as well as metabotropic receptors (P2Y). P2X receptors are involved in fast excitatory synaptic signalling by ATP, whereas the role of P2Y receptors in synaptic transmission is unclear. Seven different mammalian P2X receptor subunits (P2X1-7) have been cloned to date. This article gives an overview about the distribution of these P2X receptor subunits in the nervous system. A comparison is made between the pharmacological properties of recombinant receptors and natively occurring neuronal P2X receptors by means of electrophysiological methods. The subcellular distribution of, developmental influences on, and interspecies differences between P2X receptors are also considered. It is concluded that the properties of native P2X receptors are best explained by a heteromeric assembly of different P2X receptor subunits.     

Actions of a series of PPADS analogs at P2X1 and P2X3 receptors

Seven PPADS ( P yridoxal‐5′‐ P hosphate 6‐ A zophenyl 2′,4′‐ D i S ulfonate) analogs were investigated at Group 1 P2X receptors expressed in Xenopus oocytes. All seven analogs potently inhibited P2X₁ (IC₅₀ range, 5–32 nM) and P2X₃ (IC₅₀ range, 22–345 nM), the two Group I P2X receptor subtypes. Analogs showed greater inhibitory activity where the pyridoxal moiety of PPADS contained a 5′‐phosphonate group, rather than a 5′‐phosphate group. Analogs also showed greater potency where disulfonate groups were removed from, or substituted at, the azophenyl moiety. The most active analog was MRS 2257 (pyridoxal‐5′‐phosphonate 6‐azophenyl 3′,5′‐bismethylenephosphonate) at P2X₁ (IC₅₀, 5 nM) and P2X₃ (IC₅₀, 22 nM) receptors, being 14‐fold and 10‐fold more potent than PPADS itself. MRS 2257 produced a nonsurmountable inhibition when tested against a range of ATP concentrations, although blockade was reversed by about 85% after 20 minutes of washout. TNP‐ATP and Ip₅I were equipotent with MRS 2257 at P2X₁ receptors, whereas TNP‐ATP was 64‐fold more potent than MRS 2257 at P2X₃ receptors. In conclusion, the PPADS template can be altered at the pyridoxal and phenyl moieties to produce P2X₁ and P2X₃ receptor antagonists showing higher potency and greater degree of reversibility than the parent compound at these Group I P2X receptors. Drug Dev. Res. 53:281–291, 2001. © 2001 Wiley‐Liss, Inc.     

Pharmacological similarities between native brain and heterologously expressed alpha4beta2 nicotinic receptors

1 We studied the pharmacological properties of native rat brain and heterologously expressed rat alpha4beta2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-alpha4 antibody mAb 299. 2 Immunodepletion with the anti-beta2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained beta2. 3 The association and dissociation rate constants for 30 pM +/-[3H]-epibatidine binding to alpha4beta2 receptors expressed in oocytes were 0.02+/-0.01 and 0.03+/-0.01 min-1 (+/-standard error, degrees of freedom=7 - 8) at 20 - 23 degrees C. 4 The Hill coefficients for +/-[3H]epibatidine binding to the native brain, alpha4beta2 receptors expressed in oocytes, and alpha4beta2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69 - 0.70 suggesting a heterogeneous receptor population. Fits of the +/-[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8 - 30 and 560 - 1,200 pM. The high-affinity sites comprised 73 - 74% of the native brain and oocyte alpha4beta2 receptor population, 85% of the CV-1 alpha4beta2 receptor population. 5 The expression of rat alpha4beta2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1. 0+/-0.2). Fits of the +/-[3H]-epibatidine binding data to a single-site model gave a KD of 40+/-3 pM. 6 DHbetaE (IC50=260-470 nM) and the novel nicotine analogue NDNI (IC50=7-10 microM) inhibited 30 pM+/-[3H]-epibatidine binding to the native brain and heterologously expressed alpha4beta2 receptors equally well. 7 The results show that alpha4beta2-containing nicotinic receptors in the rat brain and heterologously expressed rat alpha4beta2 receptors have similar affinities for +/-[3H]-epibatidine, DHbetaE, and NDNI.     

Molecular structure of P2X receptors

P2X receptors are ligand-gated ion channels that transduce many of the physiological effects of extracellular ATP. There has been a dramatic increase in awareness of these receptors over the past 5 or so years, in great part due to their molecular cloning and characterization. The availability of cDNA clones for the various subunits has led to rapid progress in identifying their tissue-specific expression, resulting in new ideas concerning the functional roles these receptors might play in physiological and pathophysiological processes. In addition, molecular approaches have yielded much information regarding the structure and function of the receptor proteins themselves. In this review we seek to review recent findings concerning the molecular determinants of receptor-channel function, with particular focus on ligand binding and gating, ion selectivity, and subunit assembly.     

Evidence for functional P2X4/P2X7 heteromeric receptors

The cytolytic ionotropic ATP receptor P2X7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X4, they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X4 and P2X7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X4 and P2X7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X7 although there was no change in overall expression. Coexpression of a dominant-negative P2X4 mutant (C353W) with P2X7, inhibited P2X7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non-dominant-negative mutant (S341W) did not. Coexpression of P2X4S341W with P2X7 produced a current that was potentiated by ivermectin and inhibited by 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X4 and P2X7, which suggests that they associate to form heteromeric receptors.     

P2X receptors and nociception.

The potential importance for nociception of P2X receptors, the ionotropic receptors activated by ATP, is underscored by the variety of pain states in which this endogenous ligand can be released. Several important findings have been made recently indicating that P2X receptors can be involved in pain mechanisms both centrally and in the periphery. The roles of ATP at these two sites and the P2X receptor subtypes involved appear to be different. In the periphery, ATP can be released as a result of tissue injury, visceral distension, or sympathetic activation and can excite nociceptive primary afferents by acting at homomeric P2X(3) or heteromeric P2X(2/3) receptors. Centrally, ATP released from central afferent terminals or second order neurons can modulate neurotransmitter release or postsynaptically activate neurons involved in central nociceptive transmission, with P2X(2), P2X(4), P2X(6), and some other receptors being potentially involved. Evidence from in vivo studies suggests that peripheral ATPergic mechanisms are most important under conditions of acute tissue injury and inflammation whereas the relevance of central mechanisms appears to be more limited. Furthermore, the release of ATP and P2X receptor-mediated afferent activation appear to have been implicated in visceral and neuropathic pain; the importance of the ATPergic component in these states needs to be investigated further. Thus, peripheral P2X receptors, and homomeric P2X(3) and/or heteromeric P2X(2/3) receptors in particular, constitute attractive targets for analgesic drugs. The development of selective antagonists of these receptors, suitable for a systemic in vivo use although apparently difficult, may prove a useful strategy to generate analgesics with a novel mechanism of action.     

Kinetics of antagonist actions at rat P2X2/3 heteromeric receptors

1. Currents through heteromeric P2X(2/3) receptors were evoked by applying alpha,beta-methylene-ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X(2) and P2X(3) subunits. The concentration of alpha,beta-methylene-ATP were < or =30 microM because higher concentrations can activate homomeric P2X(2) receptors. The kinetics of action of three structurally unrelated antagonists were studied; these were 2', 3'-O-(2,4,6,trinitrophenyl)-ATP (TNP-ATP), pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. The association and dissociation rate constants were determined by pre-applying the antagonist for various periods prior to the co-application of agonist and antagonist, or by changing the solution from one containing only the agonist to one containing both agonist and antagonist. The high affinity of TNP-ATP for the P2X(2/3) receptor (K(D) approximately 2 nM) results from fast binding (k(+1) approximately 100 microM(-1) s(-1)) rather than slow unbinding (k(-1) approximately 0.3 s(-1)). For suramin (K(D) approximately 1 microM) the association rate constant ( approximately 1 microM(-1) s(-1)) was 100 times slower than that of TNP-ATP but the dissociation rate constant was similar (k(-1) approximately 1 s(-1)). PPADS (K(D) approximately 0.1 microM) associated and dissociated some 100 - 10,000 times more slowly than the other antagonists.     

Inhibition by ethanol of rat P2X(4) receptors expressed in Xenopus oocytes

1. The effect of ethanol on the function of P2X(4) receptors expressed in XENOPUS: oocytes was studied using two-electrode voltage-clamp recording. 2. The amplitude of current activated by 1 microM ATP was decreased by ethanol in a concentration-dependent manner over the concentration range 1 - 500 mM. The concentration of ethanol that produced 50% inhibition (IC(50)) of current activated by 1 microM ATP was 58 mM. 3. Ethanol inhibition of ATP-activated current was not dependent on membrane potential from -60 to +20 mV, and ethanol did not change the reversal potential of ATP-activated current. 4. Ethanol, 50 mM, shifted the ATP concentration-response curve to the right, increasing the EC(50) for ATP from 9.1 to 16.0 microM, but did not reduce the maximal response to ATP. 5. The results suggest that ethanol may inhibit P2X(4) receptors by decreasing the apparent affinity of the binding site for ATP. 6. Since the P2X(4) receptor is the most abundant P2X subunit in the brain, these receptors could be important effectors of ethanol action in the central nervous system.