Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.     

γ-Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes

Equilibrium binding interactions at the gamma-aminobutyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor-Cl- ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cl- flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (KD) values for [3H]muscimol and [3H]diazepam bindings are 1.9 microM and 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36Cl- flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.     

Characterization of gamma-aminobutyric acid-benzodiazepine receptor complexes by protection against inactivation by group-specific reagents

Abstract: The chemical topography of the γ-aminobutyric acid (GABA) and benzodiazepine (BZ) receptors was investigated in a thoroughly washed cortical membrane preparation of the rat. Chemical modification by several amino- and tyrosyl-selective reagents and the protection from it by direct and allosteric ligands of the GABA-BZ receptor complex were used to identify the residues at the binding sites. Inhibition of specific GABA binding by p -diazobenzenesulfonic acid (DSA), tetrani-tromethane (TNM), and N -acetylimidazole and the selective and complete protection from it by GABA and muscimol suggest the presence of a tyrosine residue at the GABA_A site. TNM, like DSA, selectively decreased the number of the low-affinity GABA receptors, and this could be completely protected only by GABA concentrations that can saturate the low-affinity sites. TNM pre-treatment also abolished the muscimol enhancement of [³H]diazepam binding, which suggests that the low-affinity GABA receptor sites are responsible for this enhancement. Inhibition of GABA binding by pyridoxal-5-phosphate (PLP) and the selective protection by GABA and muscimol support the presence of a lysine residue at the GABA_A receptor site. Complete and selective protection from diethylpyrocarbonate (DEP) inhibition of [³H]diazepam binding by flurazepam suggests the presence of a histidine residue at the BZ site. Flurazepam selectively protected from inhibition of [³H]diazepam binding by N -bromosuccinimide and N -acetylimidazole, but not that by DSA and TNM, which does not allow a unanimous conclusion regarding the presence of tyrosine or tryptophan residues at the BZ site.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

Distribution and Pharmacological Properties of the GABAA/ Benzodiazepine/Chloride Ionophore Receptor Complex in the Brain of the Fish Anguilla anguilla

In the present study, we characterized the distribution and the pharmacological properties of the different components of the GABAA receptor complex in the brain of the eel (Anguilla anguilla). Benzodiazepine recognition sites labeled "in vitro" with [3H]flunitrazepam ([3H]FNT) were present in highest concentration in the optic lobe and in lowest concentration in the medulla oblongata and spinal cord. A similar distribution was observed in the density of gamma-[3H]aminobutyric acid ([3H]GABA) binding sites. GABA increased the binding of [3H]FNT in a concentration-dependent manner, with a maximal enhancement of 45% above the control value, and, vice versa, diazepam stimulated the binding of [3H]GABA to eel brain membrane preparations. The density of benzodiazepine and GABA recognition sites and their reciprocal regulation were similar to those observed in the rat brain. In contrast, the binding of the specific ligand for the Cl- ionophore, t-[35S]butylbicyclophosphorothionate ([35S]TBPS), to eel brain membranes was lower than that found in the rat brain. In addition, [35S]TBPS binding in eel brain was less sensitive to the inhibitory effects of GABA and muscimol and much more sensitive to the stimulatory effect of bicuculline, when compared with [35S]TBPS binding in the rat brain. Moreover, the uptake of 36Cl- into eel brain membrane vesicles was only marginally stimulated by concentrations of GABA or muscimol that significantly enhanced the 36Cl- uptake into rat brain membrane vesicles. Finally, intravenous administration of the beta-carboline inverse agonist 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (20 mg/kg) and of the chloride channel blocker pentylenetetrazole (80 mg/kg) produced convulsions in eels that were antagonized by diazepam at doses five to 20 times higher than those required to produce similar effects in rats. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Subchronic treatment with cyclosporin A decreases the binding properties of the GABAA receptor in ovariectomized rats

In this study, we investigated the effect of cyclosporin A on the binding properties of the GABAA receptor in the hippocampus, known to be responsible for the induction of seizures, to clarify the mechanism of cyclosporin A-inhibited GABA neurotransmission in ovariectomized rats, as a climacterium model. The effects of single and subchronic treatments with cyclosporin A were examined on [3H]muscimol binding to hippocampal synaptosomal membranes in sham, ovariectomized, and estradiol/ovariectomized rats. A single treatment with cyclosporin A (40 mg/kg, i.p.) failed to change [3H]muscimol binding in the 3 groups, when compared with each corresponding vehicle-treated group. Subchronic treatment with cyclosporin A (40 mg/kg, i.p., once a day for 5 days) significantly decreased the amount of [3H]muscimol binding in ovariectomized rats. However, this inhibitory effect was not observed in sham or estradiol/ovariectomized rats. These results demonstrated that the binding activity of the GABAA receptor was decreased in ovariectomized rats after subchronic cyclosporin A treatment. This study supports the hypothesis that ovariectomy elevates the susceptibility to cyclosporin A-induced convulsions by accelerating the inhibitory actions of cyclosporin A on GABA neurotransmission in the hippocampus.     

Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol

Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).     

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

Dissociation of Muscimol, SR 95531, and Strychnine from GABAA and Glycine Receptors, Respectively, Suggests Similar Cooperative Interactions

Binding to gamma-aminobutyric acid-A (GABAA) receptors was studied in synaptosomal membranes of rat brain. Dissociation of [3H]muscimol and the GABAA antagonist [3H]2-(3-carboxypropyl)-3-amino-6-p-methoxyphenylpyridazinium bromide ([3H]SR 95531) binding elicited by 100-fold dilution was accelerated by excess of GABA or SR 95531. Control dissociation might be retarded by rebinding. The contribution of a rapid first phase of dissociation of the agonist [3H]muscimol was preferentially enhanced by SR 95531. In contrast, the dissociation of [3H]SR 95531 binding was preferentially accelerated by GABA. These opposite preferential accelerations can be explained by negative heterotropic cooperativity and a reversed affinity relationship of agonists and antagonists to GABAA binding sites with different affinities. Modification of the membranes by p-diazobenzenesulfonic acid (DSA) selectively decreased the accelerating effect of GABA on the dissociation of [3H]SR 95531 binding. [3H]Strychnine binding was studied in a membrane preparation of rat spinal cord. The dissociation of the antagonist [3H]strychnine elicited by dilution was preferentially accelerated by glycine. Again, pretreatment with DSA decreased selectively this negative heterotropic (i.e., allosteric) interaction. Chemical modification by DSA might be attributed to tyrosine residues responsible for similar allosteric interactions for the GABA- and glycine-gated chloride channels.     

Glycoprotein as a Constituent of Purified γ-Aminobutyric Acid/Benzodiazepine Receptor Complex: Structures and Physiological Roles of Its Carbohydrate Chain

The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydrochloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cystine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but -SH and -S-S- groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [3H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glycopeptidase A, beta-galactosidase, and alpha-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with beta-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with beta-galactosidase and glycopeptidase A were much higher than that with alpha-mannosidase may also indicate a special importance of the beta-galactosyl residue in the inhibition of GABA receptor binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of avermectin B1a on the gamma-aminobutyric acidA receptor and chloride channels in rat brain

The interaction of avermectin B1a (AVM) with the gamma-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t-butylbicyclophosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose-dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage-dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Functional reconstitution of the bovine brain GABAA receptor from solubilized components

The GABAA/benzodiazepine receptor has been solubilized from membrane preparations of bovine cerebral cortex and has been reconstituted, in a functionally active form, into phospholipid vesicles. In preliminary experiments, the receptor was labeled with the photoactive benzodiazepine [3H]flunitrazepam prior to solubilization. A peptide of apparent molecular weight 53,500 was specifically labeled by this method, and this was used as a marker for the receptor during the reconstitution procedures. The labeled protein was solubilized with approximately 40% efficiency by 1% beta-octyl glucoside. Reconstitution was achieved by mixing the solubilized proteins with a 4:1 mixture of soybean asolectin and bovine brain phospholipids, followed by chromatography on Sephadex G-50-80 to remove detergent. The incorporation of the GABAA receptor into membrane vesicles has been verified by sucrose gradient centrifugation in which the [3H]-flunitrazepam-labeled peptide comigrated with [14C]phosphatidylcholine used as a lipid marker. Vesicles prepared without labeled markers retained the ability to bind both [3H]flunitrazepam and the GABA analogue [3H]muscimol. Furthermore, the binding parameters were very similar to those measured using native membrane preparations. A novel fluorescence technique has been used to measure chloride transport mediated by the GABAA receptor in reconstituted vesicles. Chloride influx was rapidly stimulated in the presence of micromolar concentrations of muscimol and was blocked by preincubation of the membranes with muscimol (desensitization). Flux was also blocked by pretreatment with the competitive GABAA receptor blocker bicuculline or with the noncompetitive GABAA receptor antagonist picrotoxin.     

Kinetics of [3H]muscimol binding to the GABAA receptor in bovine brain membranes

The binding of the GABA receptor agonist [3H]muscimol to membrane preparations from bovine cerebral cortex has been investigated in equilibrium and kinetic experiments. Equilibrium binding curves are biphasic and suggest that [3H]muscimol binds to both high-affinity (Kd approximately 10 nM) and low-affinity (Kd approximately 0.5 microM) sites. Binding to each class of sites is inhibited by GABA and by the specific GABAA receptor antagonist bicuculline. The kinetics of [3H]muscimol binding have been measured by using both manual filtration assays and an automated rapid filtration technique which permits the measurement of ligand dissociation on subsecond time scales. Association and dissociation curves are biphasic at all concentrations of [3H]muscimol studied, even under conditions of low receptor saturation when no significant occupancy of the low-affinity sites would be expected. These results cannot be simply explained by the presence of two populations of binding sites in the membrane preparations but suggest the existence of two forms of the monoliganded receptor. Dissociation constants for these two forms have been estimated to be 16 and 82 nM at 23 degrees C. At higher ligand concentrations, kinetic measurements have suggested that the binding of [3H]muscimol to low-affinity sites is accompanied by a slow conformational change of the receptor-ligand complex.     

Radiation Inactivation Studies of the Benzodiazepine/γ-Aminobutyric Acid/Chloride Ionophore Receptor Complex

Radiation inactivation was used to estimate the molecular weight of the benzodiazepine (BZ), gamma-aminobutyric acid (GABA), and associated chloride ionophore (picrotoxinin/barbiturate) binding sites in frozen membranes prepared from rat forebrain. The target size of the BZ recognition site (as defined by the binding of the agonists [3H]diazepam and [3H]flunitrazepam, the antagonists [3H]Ro 15-1788 and [3H]CGS 8216, and the inverse agonist [3H]ethyl-beta-carboline-3-carboxylate) averaged 51,000 +/- 2,000 daltons. The presence or absence of GABA during irradiation had no effect on the target size of the BZ recognition site. The apparent molecular weight of the GABA binding site labelled with [3H]muscimol was identical to the BZ receptor when determined under identical assay conditions. However the target size of the picrotoxinin/barbiturate binding site labelled with the cage convulsant [35S]t-butylbicyclophosphorothionate was about threefold larger (138,000 daltons). The effects of lyophilization on BZ receptor binding activity and target size analysis were also determined. A decrease in the number of BZ binding sites (Bmax) was observed in the nonirradiated, lyophilized membranes compared with frozen membranes. Lyophilization of membranes prior to irradiation at -135 degrees C or 30 degrees C resulted in a 53 and 151% increase, respectively, in the molecular weight (target size) estimates of the BZ recognition site when compared with frozen membrane preparations. Two enzymes were also added to the membrane preparations for subsequent target size analysis. In lyophilized preparations irradiated at 30 degrees C, the target size for beta-galactosidase was also increased 71% when compared with frozen membrane preparations. In contrast, the target size for glucose-6-phosphate dehydrogenase was not altered by lyophilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the GABA/benzodiazepine receptor with the arginine reagent, 2,3-butanedione

Treatment of either crude or purified preparations of the gamma-aminobutyrate (GABA)/benzodiazepine receptor complex with arginine-specific reagents resulted in a time- and concentration-dependent loss of [3H]muscimol binding activity. Following exposure to either 2,3-butanedione or phenylglyoxal (less than or equal to 20 mM), [3H]muscimol binding was inhibited by up to 80%. [3H]Flunitrazepam binding was much less sensitive to the effects of the reagents. Scatchard analysis of the binding data indicated that treatment with butanedione resulted in a loss of [3H]muscimol binding sites with little effect on binding affinity. Considerable protection against inactivation was provided by arginine and by the endogenous receptor ligand, GABA. These results indicate that arginine residues play a critical role in maintaining the GABA receptor in a conformation capable of ligand binding, possibly by participating in the binding site through interaction with the carboxylate moiety of GABA.     

Purification and Characterization of Naturally Occurring Benzodiazepine Receptor Ligands in Rat and Human Brain

Chemicals that are active at the benzodiazepine receptor (endozepines) are naturally present in the CNS. These substances are present in tissue from humans and animals and in plants and fungi. Using selective extraction protocols, HPLC purification, receptor binding displacement studies, and selective anti-benzodiazepine antibodies, we have identified six or seven peaks of endozepines in rat and human brain. All material could competitively displace [3H]flunitrazepam binding to cerebellar benzodiazepine binding sites. Two peaks also competitively displaced Ro 5-4864 binding to the mitochondrial benzodiazepine binding site. Total amounts of brain endozepines were estimated to be present in potentially physiological concentrations, based on their ability to displace [3H]flunitrazepam binding. Although endozepine peaks 1 and 2 had HPLC retention profiles similar to those of nordiazepam and diazepam, respectively, gas chromatography-mass spectrometry as well as high-performance TLC revealed biologically insignificant amounts of diazepam (less than 0.02 pg/g) and nordiazepam (less than 0.02 pg/g) in the purified material. Electrophysiologically, some purified endozepines positively modulated gamma-aminobutyric acid (GABA) action on Cl- conductance, monitored in patch-clamped cultured cortical neurons or in mammalian cells transfected with cDNA encoding various GABAA receptor subunits. These studies demonstrate that mammalian brains contain endozepines that could serve as potent endogenous positive allosteric modulators of GABAA receptors.     

Competitive Inhibition of γ-Aminobutyric Acid Receptor Binding by N-2-Hydroxyethylpiperazine-N''-2- Ethanesulfonic Acid and Related Buffers

Several Good buffers (MOPS, ACES, BES, HEPES, ADA, and PIPES) competitively inhibited both high-affinity and low-affinity [3H]gamma-aminobutyric acid receptor binding to rat brain synaptic membranes. The most potent inhibitor was MOPS, which had Ki values of 180 nM and 79 nM for the high- and low-affinity binding sites, respectively. HEPES had Ki values of 2.25 mM and 115 microM. The buffers had no appreciable effect on sodium-dependent GABA binding or on gamma-aminobutyrate aminotransferase activity. Surprisingly, the buffers were extremely ineffectual as inhibitors of either high- or low-affinity [3H]muscimol binding. Indeed, they were of the order of 10(5) times less effective in this case than against [3H]GABA binding. These results clearly show (a) that the use of such buffers as MOPS or HEPES should be avoided in studying the interaction of GABA with its receptor, and (b) the binding sites of [3H]GABA and [3H]muscimol are not identical.     

Action of tremorgenic mycotoxins on GABAA receptor

The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on gamma-aminobutyric acid (GABAA) receptor binding and function in rat brain and on binding of a voltage-operated Cl- channel in Torpedo electric organ. None of the mycotoxins had significant effect on [3H]muscimol or [3H]flunitrazepam binding to the GABAA receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced 36Cl- influx and [35S] t-butylbicyclophosphorothionate [( 35S]TBPS) binding in rat brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of [35S]TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABAA receptor function by binding close to the receptor's Cl- channel. On the voltage-operated Cl- channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of [35S]TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABAA receptor function.     

Binding of [3H]DMCM, a Convulsive Benzodiazepine Ligand, to Rat Brain Membranes: Preliminary Studies

DMCM (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) produces convulsions in mice and rats, probably by interacting with benzodiazepine (BZ) receptors. Investigation of specific binding of [3H]DMCM to rat hippocampus and cortex revealed polyphasic saturation curves, indicating a high-affinity site (KD = 0.5-0.8 nM) and a site with lower affinity (KD = 3-6 nM). BZ receptor ligands of various chemical classes, but not other agents, displace [3H]DMCM from specific binding sites--indicating that [3H]DMCM binds to BZ receptors in rat brain. The regional distribution of [3H]DMCM binding is complementary to that of the BZ1-selective radioligand [3H]PrCC. Specific binding of [3H]DMCM (0.1 nM) was reduced by gamma-aminobutyric acid (GABA) receptor agonist to approximately 20% of the control value at 37 degrees C in chloride-containing buffers; the reduction was bicuculline methiodide- and RU 5135-sensitive. The effective concentrations of 10 GABA analogues in reducing [3H]DMCM binding correlated closely to published values for their GABA receptor affinity. Specific binding of [3H]DMCM is regulated by unknown factors; e.g. enhanced binding was found by Ag+ treatment of membranes, in the presence of picrotoxinin, or by exposure to ultraviolet light in the presence of flunitrazepam. In conclusion, [3H]DMCM appears to bind to high-affinity brain BZ receptors, although the binding properties are different from those of [3H]flunitrazepam and [3H]PrCC. These differences might relate in part to subclass selectivity and in part to differences in efficacy of DMCM at BZ receptors.