Solubilization of convulsant/barbiturate binding activity on the gamma-aminobutyric acid/benzodiazepine receptor complex

Binding activity of the radioactive cage convulsant [35S]t-butylbicyclophosphorothionate was solubilized from rat brain membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio] propanesulfonate. Binding (KD = 26 nM, Bmax = 0.4 pmol/mg protein) was inhibited by picrotoxin and related convulsants and by barbiturates and related depressants that interact with gamma-aminobutyric acid and benzodiazepine receptors via the picrotoxinin binding site. The convulsant/barbiturate binding activity chromatographed on gel filtration as a single peak coinciding with the benzodiazepine/gamma-aminobutyric acid receptor protein complex.     

Polychlorinated Convulsant Insecticides Potentiate the Protective Effect of NaCl Against Heat Inactivation of [3H]Flunitrazepam Binding Sites

Six polychlorinated convulsant insecticides that potently inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes also potentiate the protective effect of NaCl (200 mM) against heat inactivation of [3H]flunitrazepam binding sites on the same membranes. Similar effects were obtained with all "cage" convulsants tested. The rank order of potencies as heat protection potentiators was similar to the rank order of potencies as inhibitors of [35S]TBPS binding (alpha-endosulfan greater than endrin greater than dieldrin greater than toxaphene greater than lindane). alpha-Endosulfan and endrin are more potent in both respects than any previously reported picrotoxin-like (cage) convulsant, but are much less toxic to mammals. The greatly reduced toxicities of alpha-endosulfan and endrin in mammals may reflect partial gamma-aminobutyric acid (GABA)-neutral properties of these compounds. Time courses of heat inactivation of [3H]flunitrazepam binding sites in the presence of 200 mM NaCl plus saturating concentrations of endrin or picrotoxin revealed monophasic components constituting about 88% of the binding sites, suggesting that virtually all [3H]flunitrazepam binding sites are coupled to picrotoxin binding sites in the GABA/benzodiazepine/picrotoxin receptor complex. Protection against heat inactivation constitutes a useful tool for characterizing the various allosterically linked binding sites in neurotransmitter receptor complexes.     

Effects of Picrotoxin Treatment on GABAA Receptor Supramolecular Complexes in Rat Brain

The effects of acute and chronic administration of a subconvulsive dose of picrotoxin on t-[35S]butylbicyclophosphorothionate ([3S]TBPS), [3H]muscimol, and [3H]flunitrazepam binding characteristics in various regions and on the convulsant potency of picrotoxin in Sprague-Dawley rats were examined. Acute administration of a subconvulsive dose of picrotoxin (3 mg/kg, i.p.) significantly increased [35S]TBPS and [3H]muscimol binding in cerebellum (CB) with no change in frontal cortex (FC). In rats treated chronically with picrotoxin (3 mg/kg, i.p., daily for 10 days), the Bmax of [35S]TBPS binding site was significantly decreased in the FC, striatum (ST), and CB with no change in KD values. Neither [3H]muscimol binding in the FC and CB nor [3H]flunitrazepam binding in the FC was affected in these rats. In addition, the potency of pentobarbital to inhibit [35S]TBPS binding in vitro was not altered following acute or chronic treatment of picrotoxin. Chronic administration of picrotoxin did not affect convulsive ED50 or LD50 of picrotoxin; however, it delayed the onset of convulsions and increased the time to death. These results suggest that treatment with picrotoxin at a subconvulsive dose for 10 days causes down-regulation of [35S]TBPS binding sites and that this down-regulation might be related, at least in part, to the decreased extent of convulsant potency of picrotoxin. In addition, the results indicate possible interaction between convulsant binding sites and GABAA receptor sites in the CB following picrotoxin treatment.     

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.     

Differential Sensitivity of "Central" and "Peripheral" Type Benzodiazepine Receptors to Phospholipase A2

The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the "central" and "peripheral" types. PLA2 (0.005-0.1 U/ml) increased [3H]flunitrazepam and [3H]3-carboethoxy-beta-carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither gamma-aminobutyric acid (GABA)-enhanced [3H]flunitrazepam binding nor [3H]Ro 15-1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)- and barbiturate (pentobarbital)-enhanced [3H]flunitrazepam binding and [35S]t-butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025-0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine-GABA receptor chloride ionophore complex, which results in a selective loss of allosteric "regulation" between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5-4864 to peripheral-type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of "central" and "peripheral" types of benzodiazepine receptors.     

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.     

Convulsant potencies of tetrazoles are highly correlated with actions on GABA/benzodiazepine/picrotoxin receptor complexes in brain

A series of tetrazole convulsants were examined for their potencies in displacing [35S]-t-butylbicyclophosphorothionate (TBPS) from the picrotoxin site on the benzodiazepine-GABA-chloride ionophore receptor complex. All of the tetrazole derivatives tested inhibited [35S]-TBPS binding from rat forebrain membranes, and except for one (undecamethylenetetrazole), had Hill coefficients near unity. Similar to other chemically unrelated convulsants the inhibition of [35S]-TBPS binding by the various tetrazole derivatives was unaffected by the addition of the bicucculine-like GABA antagonist, R 5135. To ascertain whether the inhibition of specific [35S]-TBPS binding by the tetrazole derivatives was related to their convulsant properties, we compared their in vitro potencies in displacing [35S]-TBPS binding with their minimum convulsant potencies in mice. A very good correlation was observed (r = 0.96, p less than 0.001) between their relative affinities for the [35S]-TBPS binding site and their convulsant potencies, indicating that pentamethylenetetrazol and related tetrazoles may produce their convulsant and anxiogenic actions via the GABA-benzodiazepine-chloride ionophore receptor complex.     

Solubilization and Detergent Effects on Interactions of Some Drugs and Insecticides with the t-Butylbicyclophosphorothionate Binding Site Within the γ-Aminobutyric Acid Receptor-Ionophore Complex

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine-fold by EDTA/water dialysis and 1.3- to 4.2-fold by 50 nM ketosteroid R 5135, or 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or related piperazine-N-alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one-fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water-dialyzed RBM with 20 mM CHAPS. Similar KD values (64-86 nM) are obtained for the original EDTA/water-dialyzed membranes and the CHAPS-treated and/or -solubilized preparations. The Bmax of the EDTA-treated RBM is reduced five-fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: gamma-aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS: TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R, alpha S)-cis-cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor-ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the alpha-cyano pyrethroid and the barbiturate interact with TBPS binding.     

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.     

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)     

"Peripheral-Type"Binding Sites for Benzodiazepines in Brain: Relationship to the Convulsant Actions of Ro 5–4864

Previous studies have shown that Ro 5-4864 is a potent convulsant and increases the firing rate of substantia nigra zona reticulata neurons. The pharmacologic profile of compounds that antagonize these actions suggested that the effects of Ro 5-4864 were not mediated by "brain-type" benzodiazepine receptors. We examined a number of compounds that are structurally related to Ro 5-4864 for their capacities to displace [3H]Ro 5-4864 from "peripheral-type" binding sites and their potencies as convulsants (or as antagonists of Ro 5-4864-induced convulsions). It was observed that compounds such as KW 3600 (the N-desmethyl analog of Ro 5-4864), which have very low affinities for "peripheral-type" sites, are convulsants with a potency nearly equal to that of Ro 5-4864. In contrast, compounds such as Ro 5-6900 and PK 11195, which bind with very high affinities to "peripheral-type" binding sites, are neither convulsants nor do they antagonize the convulsant actions of Ro 5-4864. Within a series of compounds that are structurally related to Ro 5-4864 there is a good correlation (r = 0.93; p less than 0.01) between their potencies as convulsants and their capacities to displace [35S]t-butylbicyclophosphorothionate from sites that may be associated with the chloride ionophore. Thus, it appears that occupation of "peripheral-type" binding sites by high-affinity ligands may not be directly involved in the convulsant actions of Ro 5-4864 and related compounds.     

Quantitative Autoradiography on [(35)S]TBPS Binding Sites of Gamma- Aminobutyric Acid(A) Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred forHigh- and Low-Alcohol Preference

It has been documented that ethanol can potentiate brain -aminobutyric acid (GABA)ergic function, and there is a close link between the GABA_A receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA_A receptors might be associated with alcohol preference. In the present study, [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) was used to localize GABA_A receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [³⁵S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [³⁵S]TBPS binding sites of GABA_A receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [³⁵S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [³⁵S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [³⁵S]TBPS binding may represent only a small spectrum of the GABA_A receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In conclusion, the present study supports the working hypothesis that GABA_A receptors are involved in alcohol preference in HAD rats.     

The Peripheral-Type Benzodiazepine Receptor Is Present in Astrocytes but Is Not a Primary Site of Action for Convulsants/Anticonvulsants

Abstract: High-affinity binding sites for [³H]PK 11195 and [³H]Ro 5-4864 with the properties of the peripheral-type benzodiazepine receptor were detected in primary cultures of both mouse neocortical and cerebellar astrocytes. The binding sites were enriched in mitochondrial fractions on differential centrifugation. An 18-kDa polypeptide was specifically photolabelled in cerebellar astrocytes by [³H]-PK 14105, a photolabel for the peripheral-type benzodiazepine receptor. However, this polypeptide did not show any reactivity with an antiserum previously raised against the corresponding polypeptide from rat adrenal gland. Various anticonvulsant and convulsant agents were tested for their ability and potency at inhibiting [³H]Ro 5-4864 binding to neocortical astrocytes. Many of these compounds, previously reported to be inhibitors of diazepam binding to neocortical astrocytes, proved ineffective in this study. No correlation was observed between convulsant/anticonvulsant potency and ability to inhibit [³H]Ro 5-4864 binding to the peripheral-type benzodiazepine receptor in these cells. Thus, whereas some convulsants and anticonvulsants might interact with this astrocytic receptor, such a system has no validity as a general screening method for these agents.     

Binding Characteristics of t-[35S]Butylbicyclophosphorothionate in Discrete Brain Regions of Rats Made Tolerant to and Dependent on Pentobarbital

The effects of acute and continuous pentobarbital administration by pellet implantation on binding characteristics of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) in discrete regions of rat brains were examined. Acute administration of pentobarbital (60 mg/kg, s.c.) affected neither the KD nor the Bmax values of [35S]TBPS binding in any of the regions studied. The cerebella of pentobarbital-tolerant rats had an increased density of [35S]TBPS binding sites with no change in their apparent affinity. There were no significant changes in the binding characteristics in the frontal cortex (FC), the striatum (ST), and the substantia nigra (SN) of these animals. Twenty-four hours after removal of the pentobarbital pellets, a significant decrease in the latency of onset of first twitch response induced by pentylenetetrazol (PTZ) (50 mg/kg, i.p.) was observed. In addition, the density of [35S]TBPS binding sites was significantly increased in the FC, the SN, and the cerebellum but not in the ST. In all brain regions studied, placebo pellet implantation and pentobarbital tolerance and dependence caused no changes in the apparent affinity of [35S]TBPS binding or the IC50 of pentobarbital for the inhibition of [35S]TBPS binding. These results suggest that [35S]TBPS binding was significantly increased following the withdrawal of the pentobarbital pellets without altering intrinsic coupling activity of barbiturate recognition sites and convulsant binding sites and that these increases in [35S]TBPS binding are related to the increased susceptibility to seizures induced by PTZ in rats made dependent on pentobarbital.     

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.     

The effects of barbiturates upon the hemodynamic responses to intravenous methionine-enkephalin in dogs: modulation by the GABA complex

In conscious animals, the intravenous administration of enkephalins increases heart rate (HR) and mean systemic arterial blood pressure (MAP); however, when given during barbiturate anesthesia, enkephalins reduce HR and MAP. We have investigated the potential role of the gamma-aminobutyric acid (GABA) complex (consisting of chloride-ion channel and binding sites for GABA, benzodiazepine, and barbiturate/picrotoxin) as the site of modulation of enkephalin responses by certain anesthetic agents in our chronically instrumented dog model. In our model, methionine-enkephalin (Met5-ENK) (35 micrograms/kg intravenously) increased HR and MAP, but following induction of general anesthesia with barbiturate (pentobarbital) or of sedation with benzodiazepine (diazepam), Met5-ENK produced vasodepressor responses despite differing levels of consciousness in the treated animals. Subsequent administration of picrotoxin restored pressor responses to Met5-ENK in the barbiturate-treated dogs, but not in those treated with benzodiazepine; picrotoxin did not alter the level of consciousness. Picrotoxin had no effect upon Met5-ENK responses in the conscious state. In contrast, alpha-chloralose, a convulsive anesthetic agent which does not appear to alter GABA complex activity, blunted but did not reverse pressor responses to Met5-ENK, despite causing a level of anesthesia similar to that produced by barbiturate. The observed pressor response to Met5-ENK during alpha-chloralose anesthesia was totally inhibited by naloxone, indicating that this response was still mediated by opiate receptors. Our data are compatible with modulation of enkephalin responses by GABA complex activity. Systemic enkephalins may generate afferent signals which may subsequently undergo GABA complex processing; the state of activation of the GABA complex may then determine whether systemic enkephalin signals are translated as vasopressor or vasodepressor responses.     

Foot-shock stress enhances the increase of [35S]TBPS binding in the rat cerebral cortex and the convulsions induced by isoniazid

We report earlier that isoniazid and foot-shock stress individually increase the maximal number of [³⁵S]TBPS binding sites (B_max) measured ex vivo in unwashed membranes from rat cerebral cortex and that the increase due to both treatments are prevented by pretreatment in vivo with diazepam which alone induced a significant decrease in the total number of [³⁵S]TBPS binding sites. In the present paper, the effect of stress was studied on both the increase in [³⁵S]TBPS binding and the convulsant activity induced by isoniazid in unstressed rats. Isoniazid induced a time dependent increase in [³⁵S]TBPS binding. The isoniazid-induced increase in [³⁵S]TBPS binding was markedly potentiated by foot-shock stress. Moreover, foot-shock stress markedly reduced the latency to the appearance of generalized seizures induced by isoniazid (300 mg/kg s.c.). The results provide evidence that the in vivo inhibition of GABAergic transmission elicited by isoniazid results in an increase of [³⁵S]TBPS binding in the rats cerebral cortex. The finding that stress, like isoniazid, enhances [³⁵S]TBPS binding suggests that this treatment also inhibits the function of GABAergic synapses.     

Autoradiographic imaging of altered synaptic alphabetagamma2 and extrasynaptic alphabeta GABAA receptors in a genetic mouse model of anxiety

To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor gamma2 subunit (gamma2+/-) were studied using ligand autoradiographic assays on brain cryostat sections. The [35S]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant alpha1/6beta3 than in alpha1/2beta3gamma2 or beta2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [35 S]TBPS binding in the gamma2+/- mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of gamma2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [3H]Ro 15-4513 autoradiography. As the gamma2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic alphabeta3 receptors can be visualized in vitro as atypical GABA-insensitive [35S]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone gamma2+/- mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.     

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.     

The Purified Gaba/Benzodiazepine/Barbiturate Receptor Complex: Four Types of Ligand-Binding Sites,and the Interactions between them,are Preserved in a Single Isolated Protein Complex

Abstract

A GABA / benzodiazepine/barbiturate receptor complex has been purified from bovine cerebral cortex by affinity chromatography on a benzodiazepine column. Depending on the detergent present during the isolation of the receptor (deoxycholate/Triton X-100 or CHAPS/Asolectin), and during the binding assays (Triton X-100 or CHAPS), the receptor displays different binding properties for the GABA_A agonist [³H]muscimol and for the chloride ion channel blocking agent [³⁵S]t-butylbicyclophosphoro-thionate (TBPS), whereas the binding properties for the benzodiazepine [³H] flunitrazepam are independent of isolation and assay conditions. Both methods of isolation yield a protein complex consisting of the same two subunits of Mr 53000 and Mr 57000. Therefore the different binding properties reflect different conformations of the isolated receptor protein. [³H] flunitrazepam binding to the CHAPS-purified receptor is stimulated by GABA and the barbiturate pentobarbital in a dose-dependent manner. Photo-affinity labeling of the purified receptor with [³H] flunitrazepam leads to incorporation of radioactivity into both subunits, but predominantly into the Mr 53000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires > 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments < M_r 10000.