Barbiturate and benzodiazepine modulation of GABA receptor binding and function

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Binding of Agonists and Antagonists to Muscarinic Acetylcholine Receptors on Intact Cultured Heart Cells

The binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured cardiac cells has been compared with the binding observed in homogenized membrane preparations. The antagonists [3H]quinuclidinyl benzilate and [3H]N-methylscopolamine bind to a single class of receptor sites on intact cells with affinities similar to those seen in membrane preparations. In contrast with the heterogeneity of agonist binding sites observed in membrane preparations, the agonist carbachol binds to a homogeneous class of low-affinity sites on intact cells with an affinity identical to that found for the low-affinity agonist site in membrane preparations in the presence of guanyl nucleotides. Kinetic studies of antagonist binding to receptors in the absence and presence of agonist did not provide evidence for the existence of a transient (greater than 30 s) high-affinity agonist site that was subsequently converted to a site of lower affinity. Nathanson N. M. Binding of agonists and antagonists to muscarinic acetylcholine receptors on intact cultured heart cells.     

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase

The binding of [³H](?)-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay. The specificity of the binding sites is strikingly similar to what might be expected of the β-adrenergic receptor binding sites which mediate stimulation of adenylate cyclase by catecholamines in these membranes. The sites bind β-adrenergic antagonists and agonists with affinities which are directly related to their antagonist or agonist potency on the frog erythrocyte membrane adenylate cyclase. Binding shows strict stereospecificity with (?)-isomers exhibiting two orders of magnitude higher affinities than (+)-isomers. Dissociation constants for potent β-adrenergic antagonists are in the range of 10^?9 – 10^?8M whereas those for β-adrenergic agonists are about two orders of magnitude higher (≥ 10^?6M).     

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.     

Diazotization and Thiocyanate Differentiate Agonists from Antagonists for the High- and Low-Affinity Receptors of γ-Aminobutyric Acid

The differentiation of high- and low-affinity postsynaptic gamma-aminobutyric acid (GABA) receptors was examined in a washed cortical membrane preparation of the rat. The selective elimination of the high- and low-affinity GABA sites by the chaotropic anion thiocyanate and diazotization by p-diazobenzenesulfonic acid (DSA), respectively, offered two model systems for the separate sites. The [3H]GABA displacing potencies of some GABA agonists [GABA, 4,5,6,7-tetrahydro- isoxazole [4,5c]pyridine-3-ol (THIP), and muscimol] and antagonists [bicuculline methiodide (BCM), 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R-5135), and d-tubocurarine] and their slope factors were examined in these model systems and in control membranes. The displacing potency of the agonists was increased in the DSA-pretreated membranes and decreased in the presence of thiocyanate. The displacing potency of the antagonists was shifted in an opposite manner. The chaotropic effect of thiocyanate was reversible and not additive with the inhibitory effect of diazotization on the specific binding of GABA. Inhibition of specific GABA binding by pyridoxal-5-phosphate (PLP) could not be protected by GABA antagonists (BCM and R-5135) but only by agonists. The results can be interpreted in the framework of a dual (agonist-antagonist) receptor model, postulating a hydrophobic accessory site at the low-affinity GABA receptor. The effect of thiocyanate on the GABA receptor may result in the exposure of the hydrophobic accessory sites.     

The GABA postsynaptic membrane receptor-ionophore complex

Summary The function of the inhibitory neurotransmitter, -aminobutyric acid (GABA), has been implicated in the mode of action of many drugs which excite or depress the central nervous system. Many convulsant agents appear to block GABA action whereas anticonvulsants enhance GABA action. Some of these drug effects involve altered GABA-mediated synaptic transmission at the level of GABA biosynthesis, release from nerve endings, uptake into cells, and metabolic degradation. A greater number of agents of diverse classes appear to affect GABA action at the postsynaptic membrane, as determined from both electrophysiological and biochemical studies. The recently developedin vitro radioactive receptor binding assays have led to a wealth of new information about GABA action and its alteration by drugs. GABA inhibitory transmission involves the regulation, by GABA binding to its receptor site, of chloride ion channels. In this GABA receptor-ionophore system, other drug receptor sites, one for benzodiazepines and one for barbiturates/picrotoxinin (and related agents) appear to form a multicomponent complex. In this complex, the drugs binding to any of the three receptor categories are visualized to have an effect on GABA-associated chloride channel regulation. Available evidence suggests that the complex mediates many of the actions of numerous excitatory and depressant drugs showing a variety of pharmacological effects.     

Evidence for Functionally Distinct Subclasses of γ-Aminobutyric Acid Receptors in Rabbit Retina

Abstract: γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian retina, where it serves many roles in establishing complex response characteristics of ganglion cells. We now provide biochemical and physiological evidence that at least three subclasses of GABA receptors (A1, A2, and B) contribute to different types of synaptic integration. Receptor binding studies indicate that approximately three-fourths of the total number of [³H]GABA binding sites in retina are displaced by the GABA_A receptor antagonist, bicuculline, whereas one-fourth are displaced by the GABA-B receptor agonist, baclofen. GABA_A receptors can be described by a three-site binding model with K_D values of 19 n M , 122 n M , and 5.7 μ M . Benzodiazepines and barbiturates potentiate binding to the GABA_A site, which suggests that significant numbers of GABA_A receptors are coupled to regulatory sites for these compounds and thus are classified as GABA_A1 receptors. The response to pentobarbital appears to involve a conversion of low-affinity sites to higher-affinity sites, and is reflected in changes in the densities of sites at different affinities. Functional studies were used to establish which of the different receptor subclasses regulate release from cholinergic amacrine cells. Our results show that GABA suppresses light-evoked [³H]acetylcholine release via GABA_A2 receptors not coupled to a benzodiazepine or barbiturate regulatory site, and enhances release via GABA_B receptors. GABA_A1 sites do not appear to control acetylcholine release in rabbit retina.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.     

The GABA hypothesis of the pathogenesis of hepatic encephalopathy: current status

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter of the mammalian brain, can induce coma. Outside the central nervous system it is synthesized by gut bacteria and catabolized largely in the liver. GABA and its agonists, as well as benzodiazepines and barbiturates, induce neural inhibition as a consequence of their interaction with specific binding sites for each of these classes of neuroactive substances on the GABA receptor complex of postsynaptic neurons. In a rabbit model of acute liver failure: (i) the pattern of postsynaptic neuronal activity in hepatic coma, as assessed by visual evoked potentials, is identical to that associated with coma induced by drugs which activate the GABA neurotransmitter system (benzodiazepines, barbiturates, and GABA agonists); (ii) the levels of GABA-like activity in peripheral blood plasma increase appreciably before the onset of hepatic encephalopathy, due at least in part to impaired hepatic extraction of gut-derived GABA from portal venous blood; (iii) the blood-brain barrier becomes abnormally permeable to an isomer of GABA, alpha-amino-isobutyric acid, before the onset of hepatic encephalopathy; and (iv) hepatic coma is associated with an increase in the density of receptors for GABA and benzodiazepines in the brain. These findings are the bases of the following hypotheses: (i) when the liver fails, gut-derived GABA in plasma crosses an abnormally permeable blood-brain barrier and by mediating neural inhibition contributes to hepatic encephalopathy; (ii) an increased number of GABA receptors in the brain found in liver failure increases the sensitivity of the brain to GABA-ergic neural inhibition; and (iii) an increased number of drug binding sites mediates the increased sensitivity to benzodiazepines and barbiturates observed in liver failure by permitting increased drug effect.     

Beta-adrenergic receptor in the brain: comparison of 3H-dihydroalprenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in cell free homogenates.

The properties of specific ³H-dihydroalprenolol binding sites resemble the properties of the beta-receptor regulating hormone-sensitive adenylyl cyclase activity in an homogenate of rabbit cerebellum. The rabbit cerebellum has 5 to 6 pmole per gm (wet weight) of high affinity (K_D=1.3 nM) specific binding sites for ³H-dihydroalprenolol. the interaction of several beta-adrenergic agonists and antagonists with the specific binding sites is rapid, reversible, and demonstrates stereospecificity which parallels the properties of the beta receptor. Beta-adrenergic agonists show a similar potency as agonists upon adenylyl cyclase activity and as inhibitors of ³H-dihydroalprenolol binding: i.e. l-isoproterenol > l-epinephrine > l-norepinephrine (suggesting a beta₂ adrenergic receptor). The binding affinities of several beta-adrenergic agonists and antagonists for the specific binding sites approximate the affinities of these compounds for the stimulation of adenylyl cyclase. Thus, the ³H-dihydroalprenolol binding sites have properties similar to the beta-adrenergic receptor regulating adenylyl cyclase activity in a rabbit cerebellar homogenate.     

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.     

Beta-Adrenergic receptors: solubilization of (-)(3H)alprenolol binding sites from frog erythrocyte membranes.

The β-adrenergic receptors ((?)[³H]alprenolol binding sites) present in a purified preparation of frog erythrocyte membranes have been solubilized with digitonin and assayed by equilibrium dialysis with (?)[³H]alprenolol. At a concentration of 0.5–1% the detergent solubilizes about 80% of the receptor binding activity. The soluble receptor sites are not sedimented at centrifugal forces up to 105,000 xg for two hours, pass freely through Millipore filters of 0.22 μ pore size and fractionate on Sepharose 6B gel with an apparent molecular weight of 130–150,000 in the presence of digitonin. The soluble receptor sites retain all of the binding characteristics of the membrane-bound receptors. β-adrenergic agonists and antagonists compete with (?)[³H]alprenolol for occupancy of the soluble sites with affinities which are directly related to their β-adrenergic potency on membrane-bound adenylate cyclase.     

Solubilization by CHAPS detergent of barbiturate-enhanced benzodiazepine-GABA receptor complex

Abstract: Barbiturates enhance the binding of [³H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, K _d , for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration-dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [³H]Flunitrazepam binding activity is stable with storage at 4°C., but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly ( t _1/2= 48 h). [³H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS-solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities.     

GABA agonists

Summary This review describes the development of GABA receptor agonists with no detectable affinity for other recognition sites in GABA-mediated synapses. The key compounds are THIP, isoguvacine, and piperidine-4-sulphonic acid (P4S), developed via extensive structural modifications of the potent but not strictly specific GABA agonist muscimol. The structural parameters, which have to be considered in the design of GABA agonists are discussed on the basis of the structures and biological activities of these GABA agonists and a number of related compounds.A model, which summarizes our present knowledge of the structure of the postsynaptic GABA receptor complex, is presented, and the interaction of GABA agonists with various sites in this complex is discussed. Of particular interest are the effects of GABA agonists on the binding of diazepam to the benzodiazepine binding site, assumed to be a structural unit of the GABA receptor complex. While rigid molecules like THIP are capable of activating the GABA receptors, a certain degree of conformational mobility of GABA agonists apparently is a prerequisite for stimulation of diazepam binding in vitro at 0 °C. These findings suggest that GABA receptor functions involve conformational changes of certain elements of the receptor complex.Some aspects of the pharmacology of GABA agonists are discussed, including the attempts to develop GABA agonists with desirable pharmacokinetic and toxicological characteristics. While muscimol is a toxic compound, THIP is well tolerated by animals, and in contrast to isoguvacine, THIP penetrates into the brain after systemic administration to animals, a difference which can be explained on the basis of their protolytic properties. The attempts to develop pro-drugs of isoguvacine capable of penetrating the blood-brain barrier with subsequent decomposition in the brain tissue to isoguvacine are described.     

Muscarinic Autoreceptors of Torpedo Electric Organ Are of the M1 Subtype: Evidence by Radioligand Binding Using Selective Antagonists

The presynaptic muscarinic autoreceptor of Torpedo marmorata electric organ has been characterised by radioligand binding studies using the subtype-selective antagonists pirenzepine, (+)-telenzepine, methoctramine, and AF-DX 116. The presynaptic receptor had relatively high affinity for the M1 antagonists pirenzepine and (+)-telenzepine (Ki = 35 and 7 nM, respectively) and lower affinities for the M2 antagonists AF-DX 116 and methoctramine (Ki = 311 and 277 nM, respectively). Comparison of these binding data with those from an M2 receptor (rat heart membranes) assayed under identical conditions and with data in the recent literature suggests that the Torpedo muscarinic autoreceptor has a pharmacology most similar to the M1 pharmacological subtype of muscarinic acetylcholine receptor.     

Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors

N-[3H]Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. All agonists discern at least two classes of receptor in atrial and ventricular homogenates. The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. Atrial and ventricular affinities are in good agreement for some agonists but differ for others. Guanylyl imidodiphosphate (GMP-PNP) is without effect on the specific binding of the radioligand but alters the binding of carbachol via an apparent redistribution of receptors from one class to another; the apparent affinity at either class remains unchanged. Carbachol reveals two classes of sites in ventricular preparations, and the nucleotide mediates an interconversion from higher to lower affinity; three classes are revealed in atrial preparations, and the nucleotide eliminates the sites of highest affinity with a concomitant increase in the number of sites of lowest affinity. Taken together, the data are incompatible with the notion of different, noninterconverting sites; rather, there appear to be several possible states of affinity such that the equilibrium distribution of receptors among the various states is determined by the tissue, by the agonist, and by neurohumoral modulators such as guanylyl nucleotides. The effects of agonists and GMP-PNP cannot be rationalized in terms of a ternary complex model in which the low Hill coefficients arise from a spontaneous equilibrium between receptor (R) and G protein (G) and in which agonists bind preferentially to the RG complex.     

Inhibition of [3H] Dexetimide binding by a homologous series of methylfurthrethonium analogues at the peripheral muscarinic receptor

The interaction of a homologous series of methylfurthrethonium analogues, in which there is a gradual transition from full agonists via partial agonists to antagonists, with the peripheral muscarinic receptor was investigated by concentration-dependent inhibition of [³H] Dexetimide binding. The antagonists give normal sigmoid inhibition curves, but those of agonists follow a much flatter course. The results can be explained by assuming the presence of two non-interconverting muscarinic binding sites for which agonists have different and antagonists have identical affinities.The affinity ratio for the two binding sites decreases from 61 for methylfurthrethonium to 1 for its isopropyl analogue and parallels the decrease in intrinsic activity.     

Differential Effects of Iodide and Chloride on Allosteric Interactions of the GABAA Receptor

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.     

RO5-4864 inhibits the binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes

RO5-4864, a 1,4-benzodiazepine, has recently been shown to possess anticonvulsant, convulsant and anxiogenic properties and to inhibit Ca++-calmodulin-stimulated membrane phosphorylation. RO5-4864 inhibited the binding of [35S]t-butylbicyclophosphorothionate (TBPT) to cerebral cortex, cerebellar and hippocampus membranes, with an IC50 value of approximately 20 microM. TBPT binds apparently to the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex and appears to be a site of action for several classes of convulsant, depressant and anxiolytic drugs that modulate GABAergic transmission. RO5-4864 inhibited [35S] TBPT binding in cerebral cortex, apparently competitively. Antagonists of GABA and central benzodiazepine sites did not interfere with the ability of RO5-4864 to inhibit [35S] TBPT binding. The properties of RO5-4864 to inhibit TBPT binding are similar to other convulsants and GABA antagonists (except bicuculline) which inhibit TBPT binding. These results suggest that RO5-4864 interacts with the TBPT binding sites of the oligomeric GABA receptor complex.