Modulation of GABA Receptor Binding by Ca+2

In frozen-thawed repeatedly washed rat cortical synaptic membranes, Ca2+ (1-5 mM) decreased the binding of [3H]muscimol whereas it increased the binding of [3H]gamma-aminobutyric acid (GABA). However, the binding of [3H]GABA was decreased by the same extent as the binding of [3H]muscimol when the membranes were incubated with baclofen (a selective ligand for the GABAB binding site) and Ca2+. Scatchard analysis of [3H]muscimol binding revealed that Ca2+ reduced the density of GABA binding sites without affecting the dissociation constant. Ca2+ was more potent than Ba2+, Mg2+ was ineffective, and the Ca2+ antagonist La3+ stimulated [3H]muscimol binding. The inhibition of [3H]muscimol binding by Ca2+ was not influenced by calmodulin (50 micrograms/ml), trifluoperazine (10(-5) M), verapamil (10(-6) M), quinacrine (10(-4) M), cordycepin (0.1 mM), leupeptin (20 microM), or soybean trypsin inhibitor (0.1 mg/ml). Moreover, the effect of Ca2+ was additive to that of GABA-modulin. These results indicate that Ca2+ decreases the number of GABAA binding sites while unveiling GABAB binding sites.     

GABAA Receptor Populations Bind Agonists and Antagonists Differentially and with Opposite Affinities

Pretreatment of synaptosomal membranes with a diazo-coupling reagent and the presence of Cl- ions were used to differentiate high- and low-affinity populations of postsynaptic gamma-aminobutyric acid (GABAA) receptors. The super-low-affinity GABAA receptors were characterized by the enhancing effect of GABA on [3H]diazepam binding. The GABA antagonists 2-(3-carboxypropyl)-3-amino-4-methyl-6-phenylpyridazinium chloride (SR 95103) and 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135) shifted and suppressed the dose-response curve of GABA on diazepam binding. SR 95103 displaced the lower affinity [3H]GABA binding with higher potency. Dissociation of the binding of the antagonist 2-(3-carboxypropyl)-3-amino-6-p-methoxyphenylpyridazinium bromide ([3H]SR 95531) was polyphasic. Displacing potencies of SR 95531 and GABA were examined on the major (85%) rapid and minor slower phases of dissociation separated kinetically. The slower phase corresponded to higher affinity binding of SR 95531 which was displaced by GABA with about 10 times less potency. Photoaffinity labeling with muscimol decreased the number of [3H]muscimol binding sites by 27%. It decreased the displacing potency of GABA by 72%, but not that of bicuculline methiodide. These findings can be explained by a preferential binding of antagonists to hydrophobic accessory sites around low-affinity GABAA receptors.     

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.     

High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.     

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.     

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.     

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.     

Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.     

[3H]Muscimol Binding Site on Purified Benzodiazepine Receptor

The presence of a [3H]muscimol binding site on the purified benzodiazepine receptor was demonstrated. The purified protein was apparently homogeneous as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (stained with silver), with a molecular weight of 60,000 +/- 3000. The benzodiazepine binding sites were characterized as being of the central type and the [3H]flunitrazepam binding was enhanced by GABA. This activation was antagonized by bicuculline. [3H]Muscimol specifically binds to the benzodiazepine receptor. The Scatchard plot indicates a Kd of 23 nM and the ratio [3H]flunitrazepam/[3H]muscimol is approximately unity.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

Age-Related Loss of t-[35S]Butylbicyclophosphorothionate Binding to the γ-Aminobutyric AcidA Receptor-Coupled Chloride Ionophore in Rat Cerebral Cortex

Muscimol and t-butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the gamma-aminobutyric acidA (GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age-dependent changes in the specific binding of [3H]muscimol and [35S]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2-800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780-day-old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values. This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age-related change in the molecular (structural) organization of the GABAA receptor-chloride ionophore complex in rat cerebral cortex.     

Characterization of the Binding of [3H]SR 95531, a GABAA Antagonist, to Rat Brain Membranes

A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.     

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)     

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.     

Irreversible site-directed labeling of the 4-aminobutyrate binding site by tritiated meta-sulfonate benzene diazonium. Contribution of a nucleophilic amino acid residue of the alpha1 subunit.

Tritiated meta-sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.     

The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid.

The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM-propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics.     

Bicuculline Up-Regulation of GABAA Receptors in Rat Brain

Effects of acute and subacute administration of bicuculline on [3H]muscimol, [3H]flunitrazepam, and t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to various brain regions were studied in Sprague-Dawley rats. Acute administration of bicuculline affected neither the KD nor the Bmax of the three receptor sites. In rats treated subacutely with bicuculline (2 mg/kg, i.p., daily for 10 days), [3H]muscimol binding was increased in the frontal cortex, cerebellum, striatum, and substantia nigra. Scatchard analysis revealed that subacute treatment of rats with bicuculline resulted in a significantly lower KD of high-affinity sites in the striatum and in a significantly lower KD of high- and low-affinity sites in the frontal cortex. In the cerebellum, two binding sites were apparent in controls and acutely treated animals; however, only the high-affinity site was defined in subacutely treated animals, with an increase in the Bmax value. Triton X-100 treatment of frontal cortical membranes eliminated the difference in [3H]muscimol binding between control and subacute bicuculline treatments. On the other hand, [3H]muscimol binding was significantly increased in the cerebellum from bicuculline-treated animals even after Triton X-100 treatment. The apparent Ki of bicuculline for the GABAA receptor was also decreased in the frontal cortex and the striatum following the treatment. However, subacute administration of bicuculline affected neither the KD nor the Bmax of [3H]flunitrazepam and [35S]TBPS binding in the frontal cortex and the cerebellum. These results suggest that GABAA receptors are up-regulated after subacute administration of bicuculline, with no change in benzodiazepine and picrotoxin binding sites.     

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.     

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.     

Separate subunits for agonist and benzodiazepine binding in the gamma-aminobutyric acidA receptor oligomer

The gamma-aminobutyric acidA (GABAA) agonist muscimol can be photoactivated by 254 nm illumination to affinity label its binding site in the GABAA receptor. We have conducted this reaction on the pure receptor from bovine cerebral cortex in detergent solution, showing that [3H]muscimol can produce then a specific saturable labeling. In the detergent solution, the receptor alone is sensitive to 254 nm irradiation; this reduces the efficiency of incorporation to below that in the membranes, but the competing photoreaction with [3H]muscimol is sufficient and occurs at a representative set of the muscimol-binding sites, such that it can be employed for the photolabeling of those sites. The affinity of [3H]muscimol displayed in this irreversible reaction is indistinguishable from that of its reversible binding. gamma-Aminobutyric acid and bicuculline compete in the photolabeling reaction according to their known affinities at the gamma-aminobutyric acid-binding site. The labeling is shown to occur at the beta-subunit (apparent Mr 57,000) in the pure receptor. The binding sites for gamma-aminobutyric acid agonists, on the beta-subunits, and the benzodiazepine binding sites, on the alpha-subunits, are linked allosterically so that a strongly cooperative hetero-oligomeric structure of this receptor is deduced.