Low-affinity Gamma-aminobutyric Acid Research Articles

Effects of the crude venom of the social wasp Agelaia vicina on gamma-aminobutyric acid and glutamate uptake in synaptosomes from rat cerebral cortex.

Glutamate (L-glu) is the most important excitatory neurotransmitter in the mammalian central nervous system. Its action is terminated by transporters located in the plasma membrane of neurons and glial cells, which have a critical role in preventing glutamate excitotoxicity under normal conditions. The neurotransmitter gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Venoms of solitary wasps and orb-spiders are composed of large proteins, medium-size peptides, polyamine amides (PAs), and other neuroactive components that are highly selective to nervous tissues. The abnormal operation of uptake systems is involved in several failures. Several studies indicate alterations in extracellular GABA and glutamate concentrations in epilepsy conditions that may relate to transporter functions. The effects of the crude and boiled venom of the social wasp Agelaia vicina, "cassununga," on GABA and L-glu uptake in rat cerebral cortex synaptosomes are related. The venom uncompetitively inhibited high- and low-affinity GABA uptake by 91.2% and by 76%, respectively. This kind of inhibition was also found to affect high- (99.6%) and low-affinity (90%) uptake of L-glu. These results suggest that the effects observed in these experiments indicate the venom of A. vicina to be a useful tool to further characterize GABA- and L-glu-uptake systems.

Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium.

Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Müller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta-alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an important role in the removal of GABA from the subretinal space (SRS). This transporter could also coordinate the activities of GABA and taurine in the SRS after transitions between light and dark.

Enhancement by GABA of the association rate of picrotoxin and tert‐butylbicyclophosphorothionate to the rat cloned α1β2γ2 GABAA receptor subtype

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.

Radioautographic Evidence that the GABAA Receptor Antagonist SR 95531 is a Substrate Inhibitor of MAO‐A in the Rat and Human Locus Coeruleus

The locus coeruleus (LC), a major noradrenergic nucleus in the brain, probably has a functional role in the regulation of anxiety states as well as vigilance, attention, learning and memory. LC neurons are under the inhibitory control of gamma-aminobutyric acid (GABA) via ionotropic GABAA receptors. However, to date, little is known of the receptor binding characteristics of these neurons. In the present investigation we therefore examined by receptor radioautography the localization of the binding sites for different components of the GABAA receptor complex in the rat and human LC. Both rat and human LC neurons have a high density of binding sites for the pyridazinyl-GABA derivative [3H]SR 95531 (gabazine, a GABAA receptor antagonist for low affinity GABA recognition sites). However, at the concentrations used, no binding sites in the LC were detectable for the benzodiazepine receptor antagonist [3H]flumazenil, the GABAA receptor agonist (for high affinity sites) [3H]muscimol or the ionophore ligand [35S]t-butyl bicyclophosphorothionate (TBPS). Unexpectedly, the pharmacological specificity of [3H]SR 95531 binding to the LC differed markedly from that to most brain regions (IC50 values for GABA and RU 5135 respectively in the LC were > 10(-2) and 10(-3) M; and, for example, in the dentate gyrus the most labelled structure after the LC, 8 x 10(-7) and 1.8 x 10(-9) M). These differences prompted the further characterization of [3H]SR 95531 binding in the LC, revealing a significant affinity for monoamine oxidase type A (MAO-A), which is highly concentrated in this nucleus. In a competition binding study, a reduction of up to 25% of the [3H]SR 95531 binding was observed with MAO-A but not MAO-B inhibitors, at concentrations which produce maximum but selective enzyme inhibition. Correspondingly, 2 h after the oral administration of supramaximal doses of the MAO-A inhibitors moclobemide and Ro 41-1049 (but not the MAO-B inhibitor lazabemide) the in vitro binding of [3H]SR 95531 was markedly reduced (by 77 and 82% of controls respectively). Moreover, enzyme radioautography with [3H]Ro 41-1049 revealed that SR 95531 has a significant affinity for MAO-A (IC50 values were 10(-5) and 4 x 10(-6) M in the LC and dentate gyrus respectively) but not for MAO-B ([3H]lazabemide binding). Altogether, these findings suggest that the high-affinity binding of [3H]SR 95531 to the LC mainly reflects its affinity for MAO-A, which questions its utility as a selective ligand for low-affinity GABA recognition sites in the CNS.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain [corrected

Two novel gamma-aminobutyric acid (GABA) transporters, GAT3 and GAT4, were cloned from the mouse neonatal brain cDNA library and expressed in Xenopus oocytes. Sequence analysis indicated they were members of the Na(+)-dependent neurotransmitter transporter family. The GABA uptake activities were measured in cRNA injected Xenopus oocytes. The Km for GABA uptake by GAT3 was 18 microM and by GAT4 was 0.8 microM. GAT3 also transports beta-alanine and taurine with Km of 28 and 540 microM, respectively. Similarly, GAT4 transports beta-alanine with Km of 99 microM and taurine with a Km of 1.4 mM. The newly cloned GABA transporters were compared with two previously cloned GABA transporters, GAT1 and GAT2, in terms of molecular and pharmacological properties. While GAT1 and GAT4 gene expression were neural specific, GAT2 and GAT3 mRNAs were detected in other tissues such as liver and kidney, in which GAT3 mRNA was especially abundant. The expression of GAT3 mRNA in mouse brain is developmentally regulated, and its mRNA is abundant in neonatal brain but not in adult brain. High affinity GABA transporters GAT1 and GAT4 were more sensitive to inhibition by nipecotic acid. Low affinity GABA transporters GAT2 and GAT3 were inhibited most effectively by betaine and beta-alanine, respectively. The differential tissue distribution and distinct pharmacological properties of those four GABA transporters suggest functional specialization in the mechanisms of GABA transmission termination.

Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: An immunocytochemical study

The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However, the density of the GABAA receptors was significantly increased in the THIP-treated cultures as compared to the control cultures and this effect of THIP was particularly pronounced in the processes. GABAA receptors were occasionally observed to form 'hot spots' in process-like structures and again the frequency of these areas with an extremely high density of GABAA receptors was greatly increased in the THIP-treated cultures compared to the controls. It has thus been demonstrated that the ability of GABA agonists to induce formation of low-affinity GABA receptors can be directly visualized and quantified at the EM level using the preembedding immunogold technique. It is likely that low-affinity GABAA receptors are preferentially located in the cell processes and to a considerable extent in the form of 'hot spots'. However, these 'hot spots' also contain high-affinity receptors.

Effects of Inhibitors of Protein Synthesis and Intracellular Transport on the γ‐Aminobutyric Acid Agonist‐Induced Functional Differentiation of Cultured Cerebellar Granule Cells

The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the gamma-aminobutyric acid (GABA) agonist [4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)]-induced changes in morphological differentiation and GABA receptor expression was investigated in cultured cerebellar granule cells. After 4 days in culture the neurons were exposed to the inhibitors for 6 h in the simultaneous presence of THIP. Subsequently, cultures were either fixed for electron microscopic examination or used for preparation of membranes for [3H]GABA binding assays. In some experiments the functional activity of the newly induced low-affinity GABA receptors was assessed by investigation of the ability of GABA to inhibit neurotransmitter release from the neurons. These experiments were performed to differentiate between an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low-affinity GABA receptors. In contrast, the inhibitors of intracellular transport as well as the protease inhibitor did not affect this parameter. However, studies of effects of GABA on transmitter release from monensin-treated cultures showed that transmitter release could not be inhibited by GABA in these cells in spite of the presence of low-affinity GABA receptors in the membrane preparations. This indicates that the low-affinity receptors were not located in the plasma membrane. This is in good agreement with the corresponding morphological findings, that monensin treatment led to an intense vacuolization of the Golgi apparatus, thereby preventing intracellular transport of the newly synthesized GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-affinity gamma-aminobutyric acid transport in rat brain.

The low-affinity (Km = 100-200 microM) gamma-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporter (Km = 2-4 microM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of gamma-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 microM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one gamma-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.

II. Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex

The autoradiographic localization of subcomponents of the gamma-aminobutyric acid (GABA) receptor-chloride ionophore complex has provided insight into the distribution of this macromolecular system. GABA inhibits neurons by preferentially increasing the permeability of the affected membrane to chloride ions. This inhibition can be modified by the presence of other substances which bind to the GABA receptor complex. Autoradiographic localization of specific receptor subtypes associated with this complex has been accomplished in the central nervous system. This type of analysis has been performed on high and low affinity GABA A, benzodiazepine (BZ; both BZ 1 and BZ 2) and convulsant sites. These receptor sites are situated in distinct brain regions and co-exist in several areas. Other receptor subtypes, which may be influenced by the presence of GABA, can be analyzed for comparison in order to define regions of the brain where GABA may be exerting independent effects (i.e., those not associated with chloride channels). Microscopic localization of receptor sites indicates specific areas to investigate in further studies concerning the characterization of subcomponents of the macromolecular GABA complex associated with chloride ion channels.

V. Cerebral cortical GABA and benzodiazepine binding sites in genetically seizure prone rats

Adult male and female genetically seizure-prone rats were assesed for sound-induced seizures. Heterozygous control groups were compared with mild seizure (designated GEPR 3) and severe seizure animals (GEPR 9). Groups of animals were killed and crude synaptosome fractions (P 2) prepared from freshly dissected cerebral cortices. Binding sites for gamma-aminobutyric acid (GABA) were assessed by [ 3H]-muscimol in the absence or presence of excess GABA and/or pentobarbital. Binding sites for benzodiazepines were assessed by [ 3H]-flunitrazepam in the presence of absence of clonazepam. Compared to controls, GEPR 3 animals had a modest increase and GEPR 9 animals a larger increase in Bmax for both high and low affinity GABA sites, with no change in Kd. Chloride-dependent, barbiturate-enhanced GABA binding (increased Bmax) was observed in all conditions and groups. Likewise benzodiazepine binding (Bmax) increased slightly in GEPR 9 animals. There were no observed changes in binding sites for a survey of biogenic amines. Seizure-prone animals appear to have compensatory denervation-like supersensitivity for their most prominent inhibitory receptor, which may or may not be linked to the seizure event.

Isothiouronium compounds as gamma-aminobutyric acid agonists.

Analogues of gamma-aminobutyric acid (GABA) incorporating an isothiouronium salt as a replacement for a protonated amino functional group have been investigated for activity on: GABA receptors in the guinea-pig ileum; [3H]-GABA and [3H]-diazepam binding to rat brain membranes; and GABA uptake and transamination. For the homologous series of omega-isothiouronium alkanoic acids, maximum GABA-mimetic activity was found at 3-[(aminoiminomethyl)thio]propanoic acid. Introduction of unsaturation into this compound gave two isomeric conformationally restricted analogues. The trans isomer was inactive at GABA receptors while the cis compound ((Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA)) was more potent than muscimol and GABA as a GABA agonist with respect to low affinity GABA receptor sites. Both isomers were moderately potent at inhibiting the uptake of [3H]-GABA into rat brain slices. Comparison of possible conformations of the two unsaturated isomers by interactive computer graphics modelling and comparison with muscimol has led to a plausible active conformation of ZAPA, which may be a selective and potent agonist for low affinity GABA binding sites.

GABA induces functionally active low-affinity GABA receptors on cultured cerebellar granule cells.

The effect of gamma-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 microM GABA in the culture media led to the induction of low-affinity GABA receptors (KD 546 +/- 117 nM) in addition to the high-affinity receptors (KD 7 +/- 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED50 10 +/- 3 microM) only when the cells had been cultured in the presence of 50 microM GABA, 50 microM muscimol, or 150 microM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 microM bicuculline and mimicked by 50 microM muscimol or 150 microM THIP whereas 150 microM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABAA receptors.

Diazotization and thiocyanate differentiate agonists from antagonists for the high- and low-affinity receptors of gamma-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.

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

The chemical topography of the gamma-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), tetranitromethane (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 GABAA 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 pretreatment 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 GABAA 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.

Autoradiographic localization of 3H-GABA uptake in the thyroid gland of the rat.

The uptake of gamma-aminobutyric acid (GABA) in the thyroid gland of the rat was studied autoradiographically following in vitro incubation. High-affinity GABA uptake was localized in follicle cells, whereas C cells (parafollicular cells) in general did not accumulate GABA by high-affinity transport. The follicle cells were also the main sites of low-affinity GABA uptake. Additionally, some nerve fibres were found to accumulate GABA. The predominant localization of GABA uptake in follicle cells is discussed in view of a presumed role of GABA in thyroid function.

Biochemical identification of pharmacologically and functionally distinct GABA receptors in rat brain

Receptor binding studies were undertaken in an attempt to identify and characterize pharmacologically and functionally distinct receptor sites for gamma-aminobutyric acid (GABA) in rat brain. The results indicated that the potency of bicuculline, a GABA receptor antagonist, to displace membrane-bound [3H]GABA varies significantly among different brain regions, with the greatest potency found in the cerebral cortex and midbrain. In addition, in the presence of 50 mM ammonium thiocyanate, the potency of bicuculline to displace specifically bound [3H]GABA was increased significantly, with the magnitude of this increase being greater in some brain areas than others. The biological relevance of this thiocyanate-induced shift in the potency of bicuculline to inhibit [3H]GABA binding was indicated by the finding that ammonium thiocyanate also increased the potency of bicuculline to inhibit GABA-activated benzodiazepine receptor binding, a biochemical measure of GABA receptor function. Receptor site saturation analysis revealed that ammonium thiocyanate selectively abolished the high affinity GABA binding site without affecting either the low affinity component or GABA-activated benzodiazepine receptor binding. These findings provide further evidence for the existence of pharmacologically distinct GABA receptor sites, with some being more sensitive to the blocking action of bicuculline than others. Furthermore, the data provide direct evidence to support the hypothesis that only low affinity GABA receptor sites are linked to the benzodiazepine receptor, indicating that the kinetically different GABA binding sites are also functionally distinct. The discovery that ammonium thiocyanate selectively destroys high affinity GABA receptor binding may be useful for further defining the pharmacological, biochemical, and functional differences between GABA receptors in brain.

The effects of acute and chronic ethanol administration and its withdrawal on gamma-aminobutyric acid receptor binding in rat brain.

1 The effects of acute and chronic ethanol administration, and withdrawal on the binding of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), was investigated in rat brain. 2 Acute ethanol (2 to 4 g/kg i.p. 30 min before removal of brain) produced an increase in the binding capacity of the low affinity GABA receptor binding site. 3 Following chronic ethanol administration (1 to 21 days), the GABA receptor binding characteristics were not altered. These results suggest a possible adaptation of GABA receptors to the continuous presence of ethanol at the GABA synapse. 4 During ethanol withdrawal, the affinity of the low affinity GABA receptor binding site was significantly lower than pair-fed controls at 8 and 16 h withdrawal. 5 These results suggest that GABA receptor sensitivity may play a role in some of the neuropharmacological effects of ethanol and in its withdrawal symptoms.