Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA_A receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence.
2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response.
3. These functional characteristics were monitored with Cl⁻ currents measurements in cloned rat αxβ2γ2 subtypes of GABA_A receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA_A receptors with binding of [³⁵S]-TBPS, which is a high affinity ligand specific for GABA_A receptors with exquisite sensitivity to allosteric modulations.
4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

     

Interaction of caffeine with the GABAA receptor complex: alterations in receptor function but not ligand binding

Behavioral and neurochemical evidence indicates interactions between caffeine and other adenosine receptor ligands and the gamma-aminobutyric acid (GABA)-benzodiazepine system. To assess the effects of caffeine on binding and function at the GABAA receptor, we studied the effects of behaviorally-active doses of caffeine on benzodiazepine and Cl- channel binding and on overall function of the GABAA receptor as measured by Cl- uptake. There was no effect of caffeine on benzodiazepine receptor binding in cortical synaptosomal membranes at concentrations of 1-100 microM. No effects on benzodiazepine binding were found ex vivo in mice treated with caffeine, 20 and 40 mg/kg. At the putative Cl- channel site labeled by t-butylbicyclophosphorothionate (TBPS), binding was unchanged in vitro after caffeine treatment (1 and 10 microM) in washed and unwashed membranes. However, in ex vivo studies caffeine (20 and 40 mg/kg) increased numbers of TBPS sites in unwashed but not washed membranes. Muscimol-stimulated Cl- uptake into cortical synaptoneurosomes was decreased in mice treated with caffeine, 20 and 40 mg/kg. Similar results were observed in in vitro preparations treated with 50 microM but not 100 microM caffeine. These results indicate that caffeine administration significantly alters the Cl- transport function of the GABAA receptor complex.     

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes of cloned GABAA receptors.

1. In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the alpha 1 beta 2 gamma 2 and the alpha 6 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2. The prototype compounds, U-85575 (12-chloro-5-(5-cyclopropyl-1'',2'',4''- oxadiazol-3''-yl)-2,3-dihydro-diimidazo [1,5-a;1,2-c]quinazoline), and U-92330 (5-acetyl-3-(5''-cyclopropyl-1'',2'',4''-oxadiazole-3''-yl)-7-chloro-4,5-d ihy dro [1,5-a]quinoxaline), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA-mediated Cl- currents in both subtypes were sensitive to Ro 15-1788, a classical benzodiazepine antagonist. 3. Minor changes in the ring substituents of the drugs reduced their affinity to the alpha 6 beta 2 gamma 2 subtype more pronouncedly than to the alpha 1 beta 2 gamma 2 subtype. The diimidazoquinazoline containing a 2-methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the alpha 6 beta 2 gamma 2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5-benzoyl group showed a lower affinity to the alpha 6 beta 2 gamma 2 subtype than the 5-acetyl counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of molecular pharmacophore/receptor models for GABAA/BzR subtypes: binding affinities of symmetrically substituted pyrazolo[4,3-c]quinolin-3-ones at recombinant alpha x beta 3 gamma 2 subtypes and quantitative structure-activity relationship studies via a comparative molecular field analysis.

A series of symmetrically substituted pyrazoloquinolinones was synthesized to probe the BzR binding site of different GABAA/Bz receptor subtypes. The affinities of the ligands for different BzR subtypes have been determined by radioligand binding assays on 5 distinct recombinant GABAA receptor isoforms [alpha x beta 3 gamma 2 (x = 1,2,3,5, or 6)]. Most of the ligands synthesized exhibited potent biological activity in vitro. Among them, 3 ligands exhibited enhanced affinity for the alpha 2 beta 3 gamma 2 subtype in comparison to the other subtypes, six ligands demonstrated higher affinity for the alpha 3 beta 3 gamma 2 subtype, while 2 ligands showed some enhanced affinity for the alpha 5 beta 3 gamma 2 subtype. The remainder of the ligands exhibited relatively higher affinities at the alpha 1 containing subtype. To map out the steric and electronic differences between the benzodiazepine binding subtypes, a QSAR analysis by the method of Comparative Molecular Field Analysis (CoMFA) of each receptor subtypes was carried out.     

Functional coupling of gamma-aminobutyric acid (GABA)A and benzodiazepine type II receptors: analysis using purified GABA/benzodiazepine receptor complex from bovine cerebral cortex

Possible functional coupling between gamma-aminobutyric acid (GABA) and benzodiazepine receptors was examined using a purified GABA/benzodiazepine receptor complex. The purified receptor complex was obtained by 1012-S-acetamide adipic hydrazide Sepharose 4B affinity column chromatography, following the solubilization of synaptic membrane from the bovine cerebral cortex with Nonidet P-40. The binding of [3H]GABA to the purified GABA receptor was displaced significantly by muscimol and bicuculline, GABAA receptor agonists and antagonists, respectively, but not by baclofen, a GABAB receptor agonist. On the other hand, the binding of [3H]flunitrazepam to the purified benzodiazepine receptor was found to be displaced by microM ranges of CL 218,872, which is known to be sensitive to the benzodiazepine type II receptor. Furthermore, it was found that the binding of [3H]muscimol to these purified GABAA receptors was enhanced by benzodiazepines, while the binding of [3H]flunitrazepam to these benzodiazepine type II receptors was increased by GABA receptor agonists. These enhancements by GABA agonists and benzodiazepine agonists were found to be blocked by bicuculline and a benzodiazepine receptor antagonist, Ro15-1788, respectively. These results strongly suggest that the purified receptor may consist of GABAA and benzodiazepine type II receptors and possess a functional coupling of these sites, as shown in cerebral synaptic membranes.     

'Peripheral' and 'central' type benzodiazepine receptors in Maudsley rats

In this study the binding to 'central' and 'peripheral' benzodiazepine receptors in various brain areas in MR/N and MNR/N rat strains was investigated. The specific ligands [3H]BCCE and [3H]RO 5-4864 were used for binding to 'central' and 'peripheral' benzodiazepine receptors, respectively. Neither [3H]BCCE nor [3H]RO 5-4864 binding was changed in MNR/N strain in comparison to MR/N rat strain in cerebral cortex, cerebellum, hippocampus and medulla-pons. Scatchard analysis of [3H]BCCE or [3H]RO 5-4864 binding to cerebellar membranes also revealed no changes in either affinity or density in both types of benzodiazepine receptors. Autoradiographic analysis of brain regions using microdensitometry shows no differences in [3H]flunitrazepam binding between the MR/N and MNR/N strains. Our results, therefore, fail to confirm a previous report showing alterations in benzodiazepine receptors in Maudsley rats.     

GABAA receptors: ligand-gated Cl- ion channels modulated by multiple drug-binding sites.

GABAA receptors are ligand-gated Cl- ion channels and the site of action of a variety of pharmacologically and clinically important drugs. In this review evidence is summarized indicating that these drugs, by interacting with several distinct binding sites at these receptors, allosterically modulate GABA-induced Cl- ion flux. Other results indicate that the affinity, as well as the modulatory efficacy of drugs, changes with receptor composition. A though investigation of the pharmacological properties of the individual binding sites on different GABAA receptor subtypes could open new avenues for selective modulation of GABAA receptors in different brain regions.     

Characterization of U-97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes.

1. U-97775 (tert-butyl 7-chloro-4,5-dihydro-5-[(1-(3,4,5-trimethyl)piperazino)carbonyl]- imidazo[1,5-a])quinoxaline-3-carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2. The drug produced a bell-shaped dose-response profile in the alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM GABA response (control). An agonist interaction of U-97775 with the benzodiazepine site is suggested, because Ro 15-1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]-flunitrazepam binding was inhibited by U-97775 with a Ki of 1.2 nM. 3. The enhancement of GABA currents progressively disappeared as the U-97775 concentration was raised above 1 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) was not affected by Ro 15-1788. It appears that U-97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4. U-97775 at low concentrations reduced and at high concentrations enhanced [35S]-TBPS binding. Ro 15-1788 selectively blocked the effect of U-97775 at low concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of GABAergic transmission by zolpidem, an imidazopyridine with preferential affinity for type I benzodiazepine receptors

The effect of zolpidem, an imidazopyridine derivative with high affinity at the type I benzodiazepine recognition site, on the function of the GABAA/ionophore receptor complex was studied in vitro. Zolpidem, mimicking the action of diazepam, increased [3H]GABA binding, enhanced muscimol-stimulated 36Cl- uptake and reduced [35S]TBPS binding in rat cortical membrane preparations. Zolpidem was less effective than diazepam on the above parameters. Zolpidem induced a lower increase of [3H]GABA binding (23 vs. 35%) and muscimol-stimulated 36Cl- uptake (22 vs. 40%) and a smaller decrease of [35S]TBPS binding (47 vs. 77%) than diazepam. The finding that zolpidem enhanced the function of GABAergic synapses with an efficacy qualitatively and quantitatively different from that of diazepam suggests that this compound is a partial agonist at the benzodiazepine recognition site. Thus, our results are consistent with the view that the biochemical and pharmacological profile of a benzodiazepine recognition site ligand reflects its efficacy to enhance GABAergic transmission. Whether the preferential affinity of zolpidem at the type I site is involved in its atypical biochemical and pharmacological profile remains to be clarified.     

Expression and regulation of GABA receptors in the brain]

GABA receptors are classified into two receptor subtypes: GABAA and GABAB receptors. The GABAA receptor, one of the ionotropic type receptors, is formed by various subunits (alpha, beta, gamma and delta subunits) and constitutes the GABA-gated Cl- channel. The different combinations of these subunits are known to produce functionally heterogeneous GABAA receptors both pharmacologically and physiologically. On the other hand, GABAB receptor is known to be metabotropic type which is negatively coupled with adenylate cyclase and inositol phosphate turnover systems via inhibitory GTP binding protein.     

Stable expression of type I gamma-aminobutyric acidA/benzodiazepine receptors in a transfected cell line.

Expression plasmids were constructed with cDNAs encoding the rat gamma-aminobutyric acid-A (GABAA) receptor alpha 1, beta 2, and gamma 2 subunits and were cotransfected into cultured human embryonic kidney 293 cells. A single cell line (WS-1) was established after G-418 treatment and clonal selection. This cell line contained saturable, high affinity binding sites for the benzodiazepines [3H] Ro 15-4513 and [3H]flunitrazepam that were modulated by GABA. Competition experiments with benzodiazepine receptor ligands suggest a profile characteristic of native "type I" benzodiazepine receptors, because strong correlations were observed between the Ki values of these ligands in WS-1 cells and in both cerebellar homogenates (r = 0.97, p < 0.0001) and 293 cells transiently transfected with the corresponding cDNAs (r = 0.96, p < 0.001). Fluorescence intensity in WS-1 cells loaded with the Cl(-)-specific probe 6-methoxy-N-(3-sulfopropyl)-quinolinium was reliably increased by GABA. This effect was blocked by bicuculline and augmented by midazolam, consistent with the presence of GABA-gated, benzodiazepine receptor-modulated, Cl- channels. Northern blot analysis revealed the presence of mRNAs encoding alpha 1 and gamma 2 receptor subunits. Southern blot analysis confirmed genomic integration of transfected alpha 1 and gamma 2 cDNAs. The beta 2 subunit was not detected in either Northern or Southern blot analysis, indicating that a functional type I GABAA/benzodiazepine receptor complex can be constituted without a beta subunit.     

Functionally relevant gamma-aminobutyric acidA receptors: equivalence between receptor affinity (Kd) and potency (EC50)?

Many neurotransmitter receptors bind agonists with high affinity (Kd in the nanomolar range), whereas micromolar concentrations of the same agonists are required to elicit a functional effect. We have identified low affinity agonist binding sites for the gamma-amino-butyric acidA (GABAA) receptor-chloride channel under conditions normally used in 36Cl- uptake assays (a measure of receptor function). The GABAA agonist [3H]muscimol bound to a population of receptors with a Kd (2 microM) similar to its EC50 value for 36Cl- uptake. Binding was inhibited by the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and by the GABA antagonist bicuculline methiodide. A reduction in the number of [3H]muscimol binding sites (Bmax) by a thiol-modifying reagent produced a corresponding decrease in the Emax for muscimol. The benzodiazepine diazepam enhanced the potency of muscimol in ion flux experiments but did not alter the affinity of [3H]muscimol binding sites. We propose that benzodiazepines enhance GABAergic function by increasing receptor-ion channel coupling, rather than by increasing GABAA receptor affinity. These studies suggest that the study of physiologically relevant (low affinity) binding sites is necessary when examining regulation of receptors by cellular processes, drugs, and disease.     

Correlation between gamma-aminobutyric acidA receptor ligand-induced changes in t-butylbicyclophosphoro[35S]thionate binding and 36Cl- uptake in rat cerebrocortical membranes.

We have explored the functional significance of various drug-induced changes in t-[35S]butylbycyclophosporothionate (TBPS) binding to gamma-aminobutyric acidA (GABAA) receptors by comparing them with the actions of the drugs on GABA-induced 36Cl- uptake in rat cerebrocortical membrane preparations. In the presence of micromolar concentrations of GABA, various benzodiazepine receptor agonists, 3 alpha-21-dihydroxy-5 alpha-pregnan-20-one, and pentobarbital inhibited [35S]TBPS binding, whereas ethyl-beta-carboline-3carboxylate (beta-CCE), an inverse agonist, stimulated it, in general agreement with earlier reports [Mol. Pharmacol. 23:326-336 (1983); Mol. Pharmacol. 30:218-225 (1986)]. The drug-induced changes in [35S]TBPS binding, after normalization with respect to the corresponding action of diazepam, were closely related to the relative ability of the drugs to affect 36Cl- uptake, with a correlation coefficient of 0.98 and a slope of 0.85. Upon abolishment of GABA action by the use of bicuculline, however, all the tested drugs stimulated [35S]TBPS binding to various degrees, and their relative changes displayed a lower correlation coefficient of 0.69, with a slope of 2. In particular, the effects of the anesthetic steroid and pentobarbital on [35S]TBPS binding were markedly altered by GABA, which at 2 microM increased not only their maximal effects, but also their half-maximal concentrations severalfold. On the other hand, GABA did not significantly affect these parameters for diazepam under our experimental conditions. Also, the GABA-independent changes in [35S]TBPS binding produced by various benzodiazepine receptor agonists matched reasonably well the actions of the drugs on 36Cl- uptake, with a correlation coefficient of 0.85 and a slope of 1.0. These data suggest more pronounced functional coupling of the GABA sites with those for the steroid and the barbiturate, as compared with the benzodiazepine site. It appears that the degree of [35S]TBPS binding in the presence of GABA closely reflects the functional state of GABAA receptors and may be useful for characterization of allosteric interactions between various sites on the receptors.     

Site-specific acylation of GABA-gated Cl- channels: effects on 36Cl- uptake.

Radioligand binding studies indicate that p-isothiocyanato-t-butylbicycloorthobenzoate (p-NCS-TBOB) specifically acylates GABA-gated chloride channels. Preincubation of synaptoneurosomes with p-NCS-TBOB followed by washing resulted in a concentration dependent (63-500 nM) inhibition of both muscimol-stimulated chloride uptake and [355]t-butylbicyclophosphorothionate (TBPS) binding. The extent of acylation (assessed by inhibition of [35S]TBPS binding) was highly correlated (r = 0.89; p less than 0.001) with the inhibition of muscimol-stimulated Cl- uptake. Neither basal Cl- uptake nor [3H]muscimol binding to GABAA receptors were affected by p-NCS-TBOB. Preincubation with the nonacylating 'cage' convulsant t-butylbicycloorthobenzoate (500 nM) followed by washing had no effect on either muscimol-stimulated Cl- uptake or [35S]TBPS binding. These findings indicate that p-NCS-TBOB interferes with the efficacy of muscimol promoted channel openings, but does not affect the recognition qualities of GABAA receptors. p-NCS-TBOB should prove useful in electrophysiological and biochemical studies examining the properties of GABA-gated Cl- channels.     

Ultraviolet irradiation selectively disrupts the gamma-aminobutyric acid/benzodiazepine receptor-linked chloride ionophore

The ability of UV light to affect radioligand binding and 36Cl-uptake at the gamma-aminobutyric acidA (GABAA) receptor-chloride channel complex was examined. Exposure to 302 nm UV light produced a rapid (t1/2 = 4 min) reduction in [35S]t-butylbicyclo-phosphorothionate binding (assayed in the presence of 200 mM chloride) to sites associated with the GABAA receptor-coupled chloride ionophore. Saturation analysis revealed that this effect could be attributed entirely to a decrease in the maximum number of binding sites. Exposure to UV irradiation at lower (254 nm) and higher (366 nm) wavelengths also inhibited [35S]t-butylbicy-clophosphorothionate binding, but the respective rates of inactivation were 8- and 27-fold slower, compared with 302 nm. Other anion-dependent interactions at the GABAA receptor complex were disrupted in a similar manner. In the absence of permeant anion, [3H]flunitrazepam binding to benzodiazepine receptors was unaffected by 302 nm UV irradiation, whereas chloride-enhanced [3H]flunitrazepam binding was inhibited markedly. In the presence of 250-500 mM chloride, [3H]methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate binding to benzodiazepine receptors was also inhibited after UV exposure. Basal 36Cl- uptake into synaptoneurosomes was nearly doubled after 15 min of exposure to 302 nm light, whereas pentobarbital- and muscimol-stimulated 36Cl- uptake were reduced significantly. UV irradiation at 302 nm appears to disrupt selectively the anion-dependent functional interactions at the GABAA receptor complex. The apparent wavelength specificity suggests that the gating structure (channel) may contain tryptophan and/or tyrosine residues vital to the regulation of anion movement through the ionophore portion of this supramolecular receptor-ion channel complex.     

Differential potentiation of GABAA receptor function by two stereoisomers of diimidazoquinazoline analogues.

1. U-84935, diimidazo[1,5-a;1',2'-C]quinazoline,5-(5-cyclopropyl-1,2,4-oxid iazol-3yl)- 2,3-dihydro, is a ligand of high affinity for the benzodiazepine site of the GABAA receptor composed of alpha 1 beta 2 gamma 2 subunits. 2. The efficacy of its analogues was measured with their ability to potentiate GABA-mediated Cl- currents in the whole cell configuration of the patch clamp techniques in human kidney cells (A293 cells) expressing the subtype of the GABAA receptor. 3. The analogues displayed various levels of efficacy including agonists, partial agonists and antagonists without marked changes in their affinity for the receptors. 4. The major determinant of their efficacy was the spacial configuration of a methyl substituent of the C2 atom of the rigid and planar diimidazoquinazoline ring: U-90167, containing the methyl substituent projected below the plane of the ring, markedly enhanced the GABA current with a maximal potentiation of 220 +/- 25%, while its stereoisomer, U-90168, marginally increased the GABA response with a maximal potentiation of 45 +/- 10%, to which its methyl group appeared to contribute very little. 5. U-90167 potentiated the GABA response with an EC50 of 8.1 nM and a Hill coefficient of 1.1 and did not alter the reversal potential for the Cl- current. 6. From computational modelling, the sensitive methyl group of U-90167 could be assigned to the general region for the 5-phenyl group of diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulating GABA modulators

Benzodiazepines produce a broad spectrum of behavioral effects, which may be clinically desirable or undesirable, by positively or negatively modulating the effects of GABA at GABAA receptors. Over the past 20 years, much effort has been devoted towards identifying new compounds with limited undesirable effects. Most of this work has focused on developing drugs either with lower intrinsic activity than drugs such as diazepam, or with different binding profiles at subtypes of GABAA receptors. However, the benzodiazepine binding site is only one of multiple binding sites contained within the GABAA receptor complex, and other endogenous or exogenous compounds also may positively or negatively modulate the effects of GABA. Despite the availability of ligands for each of these distinct binding sites, very little research has examined the effects of GABA modulators given in combination. This may be due, in part, to the noncompelling results of those few studies which, depending on the particular drugs, have demonstrated site-selective antagonism or only additive effects, suggesting that each site modulates the effects of GABA independently. In this issue, McMahon and France challenge this view by showing that low-efficacy benzodiazepine ligands will effectively antagonize midazolam and, at the same doses, will enhance the effects of a neuroactive steroid. These studies raise interesting questions regarding the nature of the interaction between the benzodiazepine and neurosteroid binding sites on GABAA receptors.     

Characteristics of the association of brotizolam, a thieno-triazolo diazepine derivative, with the benzodiazepine receptor: a selective and high affinity ligand of the central type I benzodiazepine receptor.

Characteristics of the association of brotizolam, a thieno-triazolo diazepine derivative, to central benzodiazepine receptors were examined. Brotizolam significantly displaced the [3H]flunitrazepam and [3H]beta-carboline carboxylate ethylester bindings to crude synaptic membrane from the rat brain. This agent had the highest affinity for benzodiazepine receptors in the cerebellum, and it was found to be 2.1 times that in the spinal cord. Furthermore, a low concentration of brotizolam potentiated the GABA-stimulated 36Cl- influx into membrane vesicles. In contrast, the bindings of [3H]8-hydroxy-2-(di-n-propylamino)tetralin to 5-hydroxytryptamine1A receptors and [3H]ketanserin to 5-hydroxytryptamine2 receptors were not affected by brotizolam. The present results suggest that brotizolam may be a selective and high affinity ligand for the type I central benzodiazepine receptor. The anxiolytic and hypnotic actions of brotizolam seem to be not due to the association with 5-hydroxytryptamine receptor, but due to the activation of the GABAA receptor complex. Furthermore, the present results suggest that the lower affinity of brotizolam to benzodiazepine receptors in the spinal cord than those in the cerebellum may be related to the low muscle relaxation action of this drug.     

Interaction of beta-carboline inverse agonists for the benzodiazepine site with another site on GABAA receptors.

1. We examined the effects of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), a beta-carboline inverse agonist for the benzodiazepine site, on gamma-aminobutyric acid (GABA)-induced Cl-currents in several cloned rat GABAA receptor subtypes expressed in human embryonic kidney cells. The Cl- currents were measured in the whole cell configuration of patch clamp techniques. 2. DMCM at low concentrations (< 0.5 microM) occupying only the benzodiazepine site decreased GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes as expected from an inverse agonist, but produced no change in the alpha 6 beta 2 gamma 2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 microM) enhanced Cl- currents in all the subtypes with a half maximal concentration of 6 to 20 microM, depending on the alpha isoform. In the alpha 1 beta 2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl- currents with a half maximal concentration of 1.9 microM. 3. Ro 15-1788 (a classical benzodiazepine antagonist) had no effect on Cl- current enhancement by DMCM and, in fact, increased the current level through blocking current inhibition by DMCM via the benzodiazepine site. Also, Cl- current enhancement by pentobarbitone or by 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one was additive to that by DMCM at saturating doses. It appears that the agonist site for DMCM is distinct from those for benzodiazepines, barbiturates and neurosteroids. 4. Among beta-carboline analogues, methyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate markedly enhanced GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 subtype, while N-methyl-beta-carboline-3-carboxamide and 1-methyl-7-methoxy-3,4-dihydro-beta-carboline did not. It appears that the 3-carboxyl ester moiety is necessary for beta-carbolines to interact with a novel site on GABAA receptors as agonists.     

6,3'-Dinitroflavone is a low efficacy modulator of GABA(A) receptors

6,3'-Dinitroflavone (6,3'-DNF) is a synthetic flavone derivative that exerts anxiolytic effects in the elevated plus maze. Based on the finding that this effect is blocked by Ro15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) which is a specific antagonist at the benzodiazepine binding site of GABA(A) receptors we investigated the interaction of 6,3'-DNF with several recombinant GABA(A) receptor subtypes. Inhibition of [(3)H]flunitrazepam binding to recombinant GABA(A) receptors in transiently transfected HEK293 cells indicated that 6,3'-DNF exhibited the highest affinity for GABA(A) receptors composed of alpha1beta2gamma2 subunits and a 2-20 fold lower affinity for homologous receptors containing alpha2, alpha3, or alpha5 subunits. Two-electrode voltage-clamp experiments in Xenopus oocytes indicated that 6,3'-DNF does not induce chloride flux in the absence of GABA, but exerts low efficacy inverse agonistic modulatory effects on GABA-elicited currents in the GABA(A) receptor subtypes alpha1beta2gamma2 and alpha5beta2gamma2. In the subtypes alpha2beta2gamma2, alpha3beta2gamma2, alpha4beta2gamma2, alpha6beta2gamma2 or alpha4beta2delta and alpha4beta3delta, 6,3'-DNF exerts either none or very low efficacy positive modulatory effects. In contrast, 100 nM Ro15-1788 exhibited weak to moderate partial agonistic effects on each receptor investigated. These data indicate that Ro15-1788 only can antagonize the weak inverse agonist effects of 6,3'-DNF on alpha1beta2gamma2 and alpha5beta2gamma2 receptors, but will enhance the weak agonistic effects on the other receptor subtypes investigated. The possible mechanism of the Ro15-1788 sensitive anxiolytic effect of 6,3'-DNF is discussed.