Adenosine A2A and dopamine D2 heteromeric receptor complexes and their function

The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A -D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the I3 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of I3 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson's disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.     

Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors

ATP is the dominant messenger for astrocyte-to-astrocyte calcium-mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca(2+)](i)) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X(6) receptor, primary rat cortical astrocytes express all cloned ligand-gated P2X (i.e., P2X(1-5) and P2X(7)) and G-protein-coupled P2Y receptors (i.e., P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(12)). These cells also express the P2Y-like UDP-glucose receptor, which has been recently recognized as the P2Y(14) receptor. Single-cell image analysis showed that only some of these receptors are coupled to [Ca(2+)](i). While ATP induced rapid and transient [Ca(2+)](i) increases (counteracted by the P2 antagonists suramin, pyridoxal-phosphate-6-azophenyl-2'-4'-disulfonic acid and oxidized ATP), the P2X(1)/P2X(3) agonist alphabetameATP produced no changes. Conversely, the P2X(7) agonist BzATP markedly increased [Ca(2+)](i); the presence and function of the P2X(7) receptor was also confirmed by the formation of the P2X(7) pore. ADP and 2meSADP also produced [Ca(2+)](i) increases antagonized by the P2Y(1) antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar-nucleotides were also detected, which represents the first functional response reported for the putative P2Y(14) receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP-induced calcium rises are at least mediated by P2X(7) and P2Y(1) receptors; additional receptors (i.e., P2X(2), P2X(4), P2X(5), P2Y(2), P2Y(4), and P2Y(14)) may also contribute.     

Type-1 and type-2 astrocytes are distinct targets for prostaglandins D2, E2, and F2 alpha.

Accumulating evidence has revealed that astrocytes are potential targets for various neurotransmitters. Here we investigated the effects of prostaglandins (PGs) on signal transduction in purified primary cultures of rat type-1 and type-2 astrocytes. PGF2 alpha, PGD2, and 9 alpha,11 beta-PGF2, a metabolite of PGD2 and a stereoisomer of PGF2 alpha, evoked a rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) in type-1, but not in type-2, astrocytes. STA2, a stable analogue of thromboxane A2, was less effective, and PGE2 showed little effect. The PG-induced rise in [Ca2+]i was not blocked by an antagonist of either PGD2 receptor or thromboxane A2 receptor. PGF2 alpha and 9 alpha,11 beta-PGF2 stimulated rapid formation of inositol trisphosphate followed by inositol bisphosphate and inositol monophosphate. On the other hand, PGE2 increased the intracellular level of cyclic AMP in type-2 astrocytes, rather than in type-1 astrocytes. The potency of PGs for cyclic AMP formation was in the following order: PGE2 greater than PGE1 greater than or equal to STA2 much greater than iloprost, a stable analogue of PGI2. PGD2 and PGF2 alpha had no effect on cyclic AMP formation. These results demonstrate that type-1 astrocytes preferentially express PGF2 alpha receptors, the activation of which leads to phosphoinositide metabolism and [Ca2+]i elevation, whereas type-2 astrocytes possess PGE receptors that are linked to cyclic AMP formation.     

The value of the CHA2DS2-VASc score for refining stroke risk stratification in patients with atrial fibrillation with a CHADS2 score 0-1: a nationwide cohort study

North American and European guidelines on atrial fibrillation (AF) are conflicting regarding the classification of patients at low/intermediate risk of stroke. We aimed to investigate if the CHA2DS2-VASc score improved risk stratification of AF patients with a CHADS2 score of 0-1. Using individual-level-linkage of nationwide Danish registries 1997-2008, we identified patients discharged with AF having a CHADS2 score of 0-1 and not treated with vitamin K antagonist or heparin. In patients with a CHADS2 score of 0, 1, and 0-1, rates of stroke/ thromboembolism were determined according to CHA2DS2-VASc score, and the risk associated with increasing CHA2DS2-VASc score was estimated in Cox regression models adjusted for year of inclusion and antiplatelet therapy. The value of adding the extra CHA2DS2-VASc risk factors to the CHADS2 score was evaluated by c-statistics, Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI). We included 47,576 patients with a CHADS2 score of 0-1, from these 7,536 (15.8%) were CHA2DS2-VASc score=0, 10,062 (21.2%) were CHA2DS2-VASc score=1, 14,310 (30.1%) were CHA2DS2-VASc score=2, 14,188 (29.8%) were CHA2DS2-VASc score=3, and 1,480 (3.1%) were CHA2DS2-VASc score=4. Of the cohort with a CHADS2 score of 0-1, the stroke/thromboembolism rate per 100 person-years increased with increasing CHA2DS2-VASc score (95% confidence interval): 0.84 (0.65-1.08), 1.79 (1.53-2.09), 3.67 (3.34-4.03), 5.75 (5.33-6.21), and 8.18 (6.68-10.02) at one year follow-up with CHA2DS2-VASc scores of 0, 1, 2, 3, and 4, respectively. Patients with a CHADS2 score=0 were not all 'low risk', with one-year event rates ranging from 0.84 (CHA2DS2-VASc score=0) to 3.2 (CHA2DS2-VASc score=3). Results from Cox regression analyses, NRI, and IDI confirmed the improved predictive ability of the CHA2DS2-VASc score in the AF patients who have a CHADS2 score of 0-1. In conclusion, the CHA2DS2-VASc provides critical information on risk of stroke in AF patients with a CHADS2 score of 0-1 that can aid a decision of using anticoagulation. Even in patients categorised as 'low risk' using a CHADS2 score=0, the CHA2DS2-VASc score significantly improved the predictive value of the CHADS2 score alone and a CHA2DS2-VASc score=0 could clearly identify 'truly low risk' subjects. Use of the CHA2DS2-VASc score would significantly improve classification of AF patients at low and intermediate risk of stroke, compared to the commonly used CHADS2 score.     

Reversible inhibition of hydrogen peroxide elimination by calcium in brain mitochondria

In the present work, the Ca(2+) dependence of mitochondrial H(2) O(2) elimination was investigated. Mitochondria isolated from guinea pig brain were energized by glutamate and malate and incubated with micromolar concentrations of Ca(2+) in the presence of ADP, preventing permeability transition pore formation. After the completion of Ca(2+) uptake, mitochondria were challenged with H(2) O(2) (5 μM), then at various time points residual H(2) O(2) was determined using the Amplex red method and compared with that in mitochondria incubated with H(2) O(2) without Ca(2+) addition. Dose-dependent inhibition of H(2) O(2) elimination by Ca(2+) was detected, which was prevented by the Ca(2+) -uptake inhibitor Ru 360. Stimulation of Ca(2+) release from Ca(2+) -loaded mitochondria by a combined addition of Ru 360 and Na(+) decreased the Ca(2+) -evoked inhibition of H(2) O(2) removal. After Ca(2+) uptake (50 μM), mitochondrial aconitase activity was found to be decreased, which was partially attributable to the impaired elimination of endogenously produced reactive oxygen species. We found that the effects of Ca(2+) and H(2) O(2) on the activity of aconitase were additive. These results confirm that Ca(2+) inhibits elimination of H(2) O(2) in mitochondria and demonstrate that this effect is concentration dependent and reversible. The phenomenon described here can play a role in the modulation of ROS handling under conditions involving excessive cellular Ca(2+) load.     

Trafficking of adenosine A2A and dopamine D2 receptors

An interaction between adenosine A2A and dopamine D2 receptors has been demonstrated previously. It is generally found that agonist treatment internalizes receptors, including A2A and D2, whereas less is known of the long-term effects involved in the agonist-mediated trafficking of A2A and D2 receptors. Furthermore, the possible influence of the antagonists on receptor trafficking is still undefined. The present studies focus on the long-term effects of A2A and D2 agonist and D2 antagonist treatments on both A2A and D2 receptor trafficking studied at three different time intervals--3, 15, and 24 h. In addition, with the fluorescence resonance energy transfer technique, formation of heteromeric A2A and D2 receptor complexes was shown in the cotransfected CHO cell line. Confocal microscopy analysis showed that a 3-h treatment with the D2 agonist induced coaggregation of A2A/D2 receptors. These A2A/D2 receptor coaggregates internalized after 15 h with a recruitment of the receptors back to the cell membrane after 24 h. In contrast to the effects of the agonist treatment, a 3-h treatment with the D2-like antagonist raclopride increased both A2A and D2 immunoreactivity, indicating that the D2 antagonist stabilizes the D2 receptor and thereby reduces the internalization of both of the A2A and D2 receptors. Taken together, an activation of either A2A and D2 receptor or blockade of D2 receptors will cause long-lasting changes in A2A and D2 receptor trafficking.     

人脑胶质瘤MMP2/TIMP2与肿瘤血管的相关性分析

目的探讨人脑胶质瘤中的基质金属蛋白酶2(MMP2)、金属蛋白酶2组织抑制剂(TIMP2)与血管内皮生长因子(VEGF)及其受体(VEGFR)表达的相关性,分析其在肿瘤血管形成中的作用。方法应用免疫组化SP法检测40例脑胶质瘤及12例正常脑组织中MMP2、TIMP2、VEGF、VEGFR1、和VEGFR 2蛋白的表达。结果 MMP2、VEGF、VEGFR1和VEGFR2蛋白表达的免疫反应评分(IRS)在胶质瘤中明显升高,TIMP2蛋白表达的免疫反应评分(IRS)在胶质瘤中明显降低,差异有统计学意义(P0.05);胶质瘤组中MMP2与VEGFR2、TIMP2蛋白表达的免疫反应分数显著相关(r=0.396,P0.01;r=0.317,P0.05)。结论 MMP2/TIMP2可能通过影响VEGFR-2介导的信号转导途径调控胶质瘤肿瘤血管生成。     

Effects of antioxidants on calcium influx through TRPM2 channels in transfected cells activated by hydrogen peroxide

Melastatin-like transient receptor potential 2 (TRPM2) channel is a redox sensitive Ca(2+)-permeable cation channel that can be gated by H(2)O(2) binding to the channel's enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 action in response to H(2)O(2) are not understood, we examined the effects of various antioxidants on H(2)O(2)-induced TRPM2 cation channel currents in transfected Chinese hamster ovary (CHO) cells. The CHO cells were transfected with cDNA coding for TRPM2. Membrane currents were measured with the conventional whole cell patch-clamp technique. The intracellular solution contained ethylenediamine tetraacetic acid (EDTA) as a chelator for Ca(2+) and heavy metal ions instead of ethylene glycol tetraacetic acid (EGTA). Moreover, we chose an intracellular Ca(2+) concentration calculated to be in the range of 1 microM. H(2)O(2) (10 mM) was added extracellularly to the bath chamber. With these conditions, we were able to evoke TRPM2 currents consistently with H(2)O(2). We next tested whether vitamins C and E or glutathione (GSH) would prevent or attenuate the induction of TRPM2 currents by H(2)O(2) when applied extracellularly or intracellularly. Unexpectedly, administration of these antioxidants did not inhibit activation of TRPM2 by H(2)O(2). In conclusion, TRPM2 channels were constitutively activated by H(2)O(2) although we could not detect any inhibitory effect of the antioxidants on H(2)O(2)-induced TRPM2 cation channel currents in CHO cells.     

The effects of trimethyltin on the Ca+2, Mg+2 and Ca+2 + Mg+2-dependent ATPases of human neuroblastoma GM 3320

Data presented here indicate neuroblastoma GM 3320 tissue homogenates exhibit ouabain insensitive Ca+2-dependent, Mg+2-independent, Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities. Inclusion of trimethyltin in homogenate preparations of these cells appears to discriminate between these various ATPase activities. At low concentrations (25 microM), trimethyltin preferentially stimulated the Ca+2-dependent, Mg+2-independent ATPase activity while inhibiting the Ca+2 + Mg+2-ATPase activity approximately 70%. At 75 microM trimethyltin, the Ca+2 + Mg+2-dependent ATPase activity is inhibited greater than 95% while the Ca+2-dependent, Mg+2-independent activity is essentially unchanged from control activity and the Mg+2-dependent, Ca+2-independent activity is inhibited approximately 50%. At concentrations greater than 75 microM, trimethyltin significantly inhibits the Ca+2-dependent, Mg+2-independent ATPase activity. Thus, at trimethyltin concentrations of 50-75 microM, preferential inhibition of the Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities of neuroblastoma GM 3320 is achieved.     

Extracellular ATP counteracts the ERK1/2-mediated death-promoting signaling cascades in astrocytes

Oxidative stress is the main cause of neuronal death in pathological conditions. Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species, activates many intracellular signaling cascades including src family and mitogen-activated protein kinases (MAPKs), some of which are critically involved in the induction of cellular damage. We previously showed that H(2)O(2)-induced cell death in astrocytes and adenosine 5(')-triphosphate (ATP), acting on P2Y(1) receptors, had a protective effect. Here, we examined the H(2)O(2)-induced changes in intracellular signaling cascades that promote cell death in astrocytes, showing the molecular mechanisms by which the activation of P2Y(1) receptors counteracts such signals. Although H(2)O(2) activated three MAPKs including ERK1/2, p38, and JNK, only the activation of ERK1/2 participated in the H(2)O(2)-evoked cell death. H(2)O(2) induced a sustained activation of ERK1/2 mainly in the nucleus region, which was well in accordance with the H(2)O(2)-induced cell death. H(2)O(2) also activated the src tyrosine kinase family, which was an upstream signal for ERK1/2. Activation of P2Y(1) receptors by 2methylthio-ADP (2MeSADP) inhibited the H(2)O(2)-evoked activation of src tyrosine kinase, resulting in the inhibition of the phosphorylated-ERK1/2 accumulation in the nucleus. 2MeSADP enhanced the gene expression and activity of protein tyrosine phosphatase (PTP), which was responsible for the inhibition of src tyrosine kinase. Thioredoxin reductase, another cytoprotective gene we previously showed to be upregulated by 2MeSADP, also controlled the activity of PTP. Taken together, ATP, acting on P2Y(1) receptors, upregulates the PTP expression and its activity, which counteracts the H(2)O(2)-promoted death signaling cascades including ERK1/2 and its upstream signal src tyrosine kinase in astrocytes.     

Effects of manganese, calcium, magnesium and lithium on the ouabain-induced seizure

The effects of the intraventricularly administered cations (Mn2+, Ca2+, Mg2+ and Li+) against the seizure induced by ouabain (3 microgram) were investigated. Mn2+, Ca2+ and Mg2+ caused definite sedation and decreased locomotor activity. But Li+ was without significant behavioral effect at the doses applied. Among the cations used, Mn2+, Ca2+ and Mg2+ showed significant anticonvulsive effect on the ouabain-induced seizure. In comparison, on the dose and molar-to-molar basis, the potency of anticonvulsive action was in the following order: Mn2+ greater than Ca2+ greater than Mg2+. On the contrary, the higher dose of Li+ potentiated the ouabain-induced seizure. The importance of the increased Ca2+ level in the extracellular space or the inhibition of Ca2+ uptake as the anticonvulsive effect of Ca2+, Mn2+ and Mg2+ was discussed.     

P2X2 and P2X3 receptor expression in the gallbladder of the guinea pig

We investigated for the first time, the distribution pattern of P2X2 and P2X3 receptors in the gallbladder of the guinea pig using immunohistochemistry. P2X2 and P2X3 receptor-immunoreactive nerve fibers were observed within the ganglia, in the interganglionic connectives, in the muscularis and in the paravascular plexus. Immunoreactivity for P2X2 and P2X3 was also observed in most neurons in the ganglionated plexus. Double-labeling studies revealed that 58.1% of all P2X2-positive neurons and 54.3% of all P2X3-positive neurons were found to display nitric oxide synthase. Over 90% of the neurons that were immunoreactive for P2X2 and P2X3 receptor were also immunoreactive for calretinin. We also found that 30.5% of P2X2- and 32.6% of P2X3-immunoreactive neurons were also immunoreactive for vasoactive intestinal peptide. No P2X2- or P2X3- immunoreactive neurons stained for calcitonin gene-related peptide; a few calcitonin gene-related peptide-immunoreactive nerve fibers also showed immunoreactivity to P2X2 or P2X3 receptors. These results further demonstrate the neurotransmitter diversity of the nerves of the gallbladder and provide an incentive for studies of the actions of these compounds in the gallbladder wall.     

Alcohol dehydrogenase 2 genotype and allelic variants are not associated with the risk for essential tremor

OBJECTIVE: To investigate the possible association between alcohol dehydrogenase 1B, beta-polypeptide (ADH2) genotype and allelic variants and the risk for developing essential tremor (ET). METHODS: Leukocytary DNA from 204 ET patients and 200 healthy controls was studied for the genotype ADH2 and the occurrence of ADH2 allelic variants using allele-specific polymerase chain reaction amplification and MslI-restriction fragment length polymorphism's analyses. RESULTS: The frequencies of the ADH2*1/ADH2*2 genotype and of the allelic variant ADH2*2 did not differ significantly in ET patients when compared with those of the controls. The mean age at onset of ET did not differ significantly between patients with genotypes ADH2*1/ADH2*2 and ADH2*1/ADH2*1. The frequencies of the genotype ADH2*1/ADH2*2 and of the allelic variant ADH2*2 in patients with voice, tongue, and chin tremors did not differ from those of the controls, whereas patients with voice tremor showed lower frequencies of mutated genotypes and ADH2*2 alleles. The frequencies of ADH2 genotypes and ADH2 alleles did not differ significantly between patients who did not drink ethanol and those who reported improvement, no improvement, or unknown response of tremor to ethanol. CONCLUSIONS: These results suggest that ADH2 genotype and allelic variants are not associated with the risk for ET in white Spanish people.     

MMP-2-PEX与MMP-2在侵袭性垂体腺瘤中的表达及意义

目的研究基质金属蛋白酶-2在C端的血红素样结构域(MMP-2-PEX)和基质金属蛋白酶-2(MMP-2)在侵袭性垂体腺瘤中的表达及其意义。方法用逆转录酶-聚合酶链反应(RT-PCR)法检测116例垂体腺瘤标本的MMP-2-PEX及MMP-2的表达,分析其与侵袭性垂体腺瘤的关系及两者之间的相关性。结果侵袭性垂体腺瘤组MMP-2-PEX mRNA表达较非侵袭性垂体腺瘤组显著降低(P<0.01)。侵袭性垂体腺瘤组的MMP-2 mRNA表达明显高于非侵袭性垂体腺瘤组(P<0.01)。侵袭性垂体腺瘤组中MMP-2-PEX mRNA表达与MMP-2 mRNA表达呈负相关(r=-0.66,P<0.05),而非侵袭性腺垂体腺瘤组中无相关性(r=-0.22,P>0.05)。结论 MMP-2-PEX低表达及MMP-2高表达与垂体腺瘤的侵袭性密切相关,MMP-2-PEX及MMP-2可以作为垂体腺瘤侵袭性的重要参考指标。     

Neurons and glial cells differentially express P2Y receptor mRNAs in the rat dorsal root ganglion and spinal cord

We examined the precise distribution of mRNAs for six cloned rat P2Y receptor subtypes, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, and P2Y14, in the dorsal root ganglion (DRG) and spinal cord by in situ hybridization histochemistry (ISHH) with 35S-labeled riboprobes. In the DRG, P2Y1 and P2Y2 mRNAs were expressed by 15% and 24% of all neurons, respectively. Although each receptor was evenly distributed between neurofilament-positive and -negative neurons, P2Y2 was rather selectively expressed by TrkA-positive neurons. Schwann cells expressed P2Y2 mRNA, and the nonneuronal cells around the DRG neurons, perhaps the satellite cells, expressed P2Y12 and P2Y14 mRNAs. No ISHH signals for P2Y4 or P2Y6 were seen in any cellular components of the DRG. In the spinal cord, P2Y1 and P2Y4 mRNAs were expressed by some of the dorsal horn neurons, whereas the motor neurons in the ventral horn had P2Y4 and P2Y6 mRNAs. In addition, astrocytes in the gray matter had P2Y1 mRNA, and the microglia throughout the spinal cord expressed P2Y12 mRNA. P2Y14 mRNA was weakly expressed by putative microglia. These findings should provide useful information in interpreting pharmacological and electrophysiological studies in this field given the lack of highly selective antagonists for each P2Y receptor subtype.     

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ to ouabain in carotid body glomus cells of adult rabbits.

Effect of the removal of extracellular Ca2+ on the response of cytosolic concentrations of Ca2+ ([Ca2+]i) to ouabain, an Na+/K+ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca2+]i (mean+/-S.E.M.; 38+/-5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca2+]i increase was abolished by the removal of extracellular Na+. D600 (50 microM), an L-type voltage-gated Ca2+ channel antagonist, inhibited the [Ca2+]i increase by 57+/-7% (n=4). Removal of extracellular Ca2+ eliminated the [Ca2+]i increase, but subsequent washing out of ouabain in Ca2+-free solution produced a rise in [Ca2+]i (62+/-8% increase, n=6, P<0.05), referred to as a [Ca2+]i rise after Ca2+-free/ouabain. The magnitude of the [Ca2+]i rise was larger than that of ouabain-induced [Ca2+]i increase. D600 (5 microM) inhibited the [Ca2+]i rise after Ca2+-free/ouabain by 83+/-10% (n=4). These results suggest that ouabain-induced [Ca2+]i increase was due to Ca2+ entry involving L-type Ca2+ channels which could be activated by cytosolic Na+ accumulation. Ca2+ removal might modify the [Ca2+]i response, resulting in the occurrence of a rise in [Ca2+]i after Ca2+-free/ouabain which mostly involved L-type Ca2+ channels.     

Magnesium potentiation of the function of native and recombinant GABA(A) receptors.

Mg2+ decreased basal and GABA-inhibited t-butylbicyclophosphoro[35S]thionate binding to GABAA receptor ion channels in rat brain sections up to 1 mM, but increased the binding at 10 mM. The Mg2+-effect was detectable in the presence of a specific GABA site competitive antagonist. Two-electrode voltage clamp recordings of recombinant alpha1beta2gamma2S, alpha1beta2, alpha2beta2gamma2S and alpha2beta2 GABAA receptors revealed a potentiation by 0.1-1 mM Mg2+ of EC20 GABA-evoked ion currents. At 10 mM, Mg2+ decreased the currents. In the absence of GABA, Mg2+ did not evoke any currents. The results show that physiologically relevant Mg2+ concentrations affect the GABA responses on GABAA receptors in native and the main recombinant receptor subtypes, suggesting putative Mg2+ binding sites on the receptor complex.