Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

Effect of picrotoxin on benzodiazepine receptors and GABA receptors with reference to the effect of C1- ion

Picrotoxin does not by itself affect [³H] diazepam binding to synaptosomal membranes of rat cerebellum; however, picrotoxin stimulated the binding in the presence of Cl^? ion or Cl^? ion plus low concentrations of GABA. On the other hand, in the presence of GABA at concentrations higher than 1 × 10^?6 M, picrotoxin inhibited [³H]diazepam binding. This inhibition seems to be the result of reduced GABA binding, which occurred in the presence of picrotoxin and Cl^? ion. These results may indicate that benzodiazepine receptors, GABA receptors, and the Cl^? ionophore are closely associated with each other.     

Molecular dynamics simulations of GABA binding to the GABAC receptor: the role of Arg104

GABA is the major inhibitory neurotransmitter in the nervous system and acts at a variety of receptors including GABA_C receptors, which are a subclass of GABA_A receptors. Here we have used molecular dynamics simulations of GABA docked into the extracellular domain of the GABA_C receptor to explain the molecular interactions of the neurotransmitter with the residues that contribute to the binding site; in particular, we have explored the interaction of GABA with Arg¹⁰⁴. The simulations suggest that the amine group of GABA forms cation-π interactions with Tyr¹⁰² and Tyr¹⁹⁸, and hydrogen-bonds with Gln⁸³, Glu²²⁰, Ser²⁴³, and Ser¹⁶⁸, and, most prominently, with Arg¹⁰⁴. Substituting Arg¹⁰⁴ with Ala, Glu, or Lys, which experimentally disrupt GABA_C receptor function, and repeating the simulation revealed fewer and different bonding patterns with GABA, or the rapid exit of GABA from the binding pocket. The simulations therefore unveil interactions of GABA within the binding pocket, and explain experimental data, which indicate that Arg¹⁰⁴ is critical for the efficient functioning of the receptor.     

Etomidate stereospecifically stimulates forebrain,but not cerebellar, 3H-diazepam binding

³H-Diazepam binding to a total particulate fraction of rat forebrain is enhanced by (+)-etomidate and GABA, but not by (?)-etomidate. The enhancement of ³H-diazepam binding by (+)-etomidate was due to a two-fold increase in binding affinity, the maximal number of sites remained unchanged. The degree of stimulation with (+)-etomidate was higher than that obtained with GABA. THIP did not stimulate ³H-diazepam binding to forebrain, and did not reverse the enhanced binding seen with (+)-etomidate or GABA. In a synaptosomal membrane preparation of rat cerebellum, unlike (+)-etomidate, GABA and muscimol produced a marked stimulation of ³H-diazepam binding. (+)-Etomidate did not inhibit ³-muscimol binding to GABA receptors, nor did it activate or inhibit other $\text{in vitro}$ receptor binding assays. The effects of (+)-etomidate on the benzodiazepine binding are different from those of gabamimetic drugs. It is proposed that like barbiturates, (+)-etomidate may affect benzodiazepine binding by interaction with the chloride ionophore which is coupled to the GABA-receptor.     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

Vertebrate GABA receptors

Physiologic-pharmacologic studies in vivo and with tissue cultures have revealed that synaptic GABA receptors exist in the vertebrate CNS. The GABA antagonist, bicuculline, can be used to detect synaptic GABA receptors in both the presence and absence of Na⁺, even though GABA binding to cerebral subcellular fractions occurs mainly to transport (uptake) receptors in the presence of Na⁺.     

[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.     

Stimulatory and protective effects of benzodiazepines on GABA receptors labeled with [3H]muscimol

We investigated the effects of benzodiazepines on [³H]muscimol binding to rat brain membranes and on heat inactivation of GABA receptors. Scatchard analysis of [³H]muscimol binding to frozen and 0.05% Triton X-100 treated membranes revealed two components; a higher affinity (Kd=2.2 nM, Bmax=1.2 pmol/mg protein) and a lower affinity component (Kd=15.9 nM, Bmax=4.4 pmol/mg protein). Diazepam and flurazepam (3 μM) increased significantly the specific binding of 40 nM but not of 2 nM [³H]muscimol. This stimulation was attributed to an increase in the affinity of the lower affinity component for GABA receptors. The time course of heat inactivation of GABA receptors revealed rapidly and then slowly denaturating Phases. These observations would suggest that there are multiple GABA receptors with different sensitivities to the heat treatment. Diazepam depressed remarkably the slowly denaturating phase(s). After heat treatment for 50 min, the single component of GABA receptors with Kd of 14.3 nM and Bmax of 0.6 pmol/mg protein survived, whereas in the membranes preincubated with 3 μM diazepam, the Kd and Bmax of the still viable GABA receptors were 14.8 nM and 1.14 pmol/mg protein, respectively. In light of these findings, the stimulation of the lower affinity component of GABA receptors may be related to the protective effect of these drugs against heat inactivation.     

Endogenous inhibitor of GABAB and GABAA receptors

An endogenous inhibitor of γ-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG 1-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na⁺-independent [³H]GABA bindings. Scatchard plot analysis of the [³H]GABA binding to GABA “B” receptor recognition site showed this inhibitor increased the K_d value (24.1 nM to 3.6 nM) without changing the B_max. On the other hand, Scatchard plot analysis of the [³H]GABA binding to GABA “A” receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the K_d value. These results suggest that the endogenous inhibitor functions as a modulator for GABA_B and GABA_A receptors.     

Structural basis for selective GABA binding in bacterial pathogens

GABA acts as an intercellular signal in eukaryotes and as an interspecies signal in host–microbe interactions. Structural characteristics of selective eukaryotic GABA receptors and bacterial GABA sensors are unknown. Here, we identified the selective GABA‐binding protein, called Atu4243, in the plant pathogen Agrobacterium tumefaciens. A constructed atu4243 mutant was affected in GABA transport and in expression of the GABA‐regulated functions, including aggressiveness on two plant hosts and degradation of the quorum‐sensing signal. The GABA‐bound Atu4243 structure at 1.28 Å reveals that GABA adopts a conformation never observed so far and interacts with two key residues, Arg²⁰³ and Asp²²⁶ of which the role in GABA binding and GABA signalling in Agrobacterium has been validated using appropriate mutants. The conformational GABA‐analogue trans‐4‐aminocrotonic acid (TACA) antagonizes GABA activity, suggesting structural similarities between the binding sites of the bacterial sensor Atu4243 and mammalian GABA_C receptors. Exploration of genomic databases reveals Atu4243 orthologues in several pathogenic and symbiotic proteobacteria, such as Rhizobium, Azospirillum, Burkholderia and Pseudomonas. Thus, this study establishes a structural basis for selective GABA sensors and offers opportunities for deciphering the role of the GABA‐mediated communication in several host–pathogen interactions.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.     

Pipecolic acid receptors in rat cerebral cortex

Identification of [¹⁴C]pipecolic acid (PA) receptors was attempted in the solubilized membrane fraction from rat cerebral cortex. Specific binding proteins for both PA and muscimol, a potent -aminobutyric acid (GABA) agonist, were detected in the same preparation. Separation of labeled PA and GABA binding proteins by glycerol gradient centrifugation has shown labeled protein bands of similar sedimentation rates, suggesting that PA and GABA may be binding to identical proteins. It seems likely that the PA binding receptor either may possess the same sedimentation characteristics as that of the GABA receptor, or both GABA and PA which is an endogenous and weak GABA agonist may bind to the same receptor complex, if not to the same binding site.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Solubilization of the Benzodiazepine/γ-Aminobutyric Acid Receptor Complex: Comparison of the Detergents Octylglucopyranoside and 3-[(3-Cholamidopropyl)-Dimethylammonio] 1-Propanesulfonate (CHAPS)

Abstract Octylglucopyranoside (OCTG) was three times more efficient than 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulfonate (CHAPS) in solubilizing the benzodiazepine (BDZ)/γ-aminobutyric acid (GABA) receptor complex from rat cerebellar synaptic membranes. OCTG-solubilized receptor preparations had ligand binding characteristics that were significantly different from those of the CHAPS-solubilized receptors. The inclusion of phospholipids in the solubilization media improved the binding characteristics of both soluble receptor preparations and appeared absolutely necessary for the maintenance of chloride facilitation of flunitrazepam (FNZ) binding to OCTG-solubilized receptors. FNZ and ethyl-β-carboline-3-carboxylate bound to OCTG-solubilized preparations with equilibrium dissociation constants of 2.2 nM and 1.6 nM, respectively, and chloride (150 mM) and GABA (100 μM) + chloride facilitated the binding of FNZ by 15% and 55%, respectively; these ligand binding characteristics are similar to those of membrane-located BDZ receptors. Cartazolate, a pyrazolopyridine that facilitated the binding of FNZ to membrane-located and CHAPS-solubilized receptors, did not facilitate FNZ binding to OCTG-solubilized receptors. These results are discussed in terms of an interaction between the membrane lipid phosphatidylserine (PS) and cartazolate; PS appears to have the capacity to inhibit the effects of cartazolate on FNZ binding. Storage of the soluble receptor preparations for 24 h at 4° resulted in the loss of several characteristic BDZ receptor binding properties. Incorporation of the OCTG-solubilized receptor complex into liposomes prevented these losses but this procedure did not protect the CHAPS-solubilized receptors. We conclude that OCTG may have some advantages over CHAPS as the detergent of choice for the solubilization and reconstitution in liposomes of a functional BDZ/GABA receptor-chloride ionophore complex.     

Effect of thiopental on hippocampal GABA receptors in rat brain

The effect of thiopental on rat brain hippocampal GABA receptors was studied in slice preparations and membrane fractions. In slice preparations, thiopental at a concentration of 10⁻⁵ M enhanced the GABA (1−5 × 10⁻⁴M) inhibition of the field potentials evoked in pyramidal neurons by stratum radiatum stimulation. In hippocampal slices obtained from chronically barbital-administered (100 mg/kg, b.i.d., 10 days) rats, less enhancement of thiopental on GABA inhibition of the field potentials was observed. In binding experiments, two components of specific [³H]GABA binding to membrane fractions were observed. While thiopental was without effect on high-affinity sites, [³H]GABA binding with low affinity was enhanced by 80% in the presence of 10⁻⁵ M thiopental. The results are discussed in relation to the phenomena underlying chronic barbiturate administration.     

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.     

The Binding of the GAB A Agonist [3H]THIP to Rat Brain Synaptic Membranes

Abstract: THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a specific GABA agonist with potent analgesic properties. The binding of radioactive THIP to thoroughly washed, frozen, and thawed membranes isolated from rat brains has been studied at 2°C under sodium ion-free conditions and compared with the binding of [³H]GABA and [³H]piperidine-4-sulphonic acid ([³H]P4S). The best computer fits to the experimental data were in all cases attained with a receptor model based on three independent binding sites, of which only the high- and medium-affinity sites could be characterised satisfactorily. While the K_D values were found to be comparable for all three ligands employed, the density of the high-affinity binding site (B_M1) was, with the exception of the membranes from the cerebellum, considerably lower for [³H]THIP than for [³H]GABA and [³ H]P4S. The regional distribution of the GABA receptors, which bind [³H]THIP, was different from those recognizing [³H]GABA and [³H]P4S. A number of analogues, including asymmetric compounds with known configuration, were tested as inhibitors of the binding of [³H]GABA, [³H]muscimol, [³H]THIP, [³H]isoguvacine, and [³H]P4S. The concentrations of the asymmetric compounds required for the inhibition of [³H]P4S binding were much higher than those required for the displacement of [³H]GABA, [³H]muscimol, [³H]THIP, and [³H]isoguvacine. The comparable relative potencies of inhibitors do, however, indicate that all of the ligands bind to the GABA receptors.