TBPS Research Articles

Ontogeny of the GABA receptor complex in chick brain: Studies in vivo and in vitro

The ontogeny of the γ-aminobutyric acid-A (GABA A) receptor complex in the chick brain was studied by specific binding of [ 3H]muscimol, [ 3H]flunitrazepam (Flu) and [ 35S] t-butylbicyclophosphorothionate (TBPS) to isolated membranes. During development in ovo, the specific binding of muscimol and flunitrazepam increased from day 8 and reached 50% of adult levels of day 20, while a comparable level of TBPS binding was achieved by day 17. The increases in TBPS and Flu binding were reflected in B max rather than K d changes. In embryonic brain, only a low-affinity site for muscimol ( K d = 23 nM) was observed while an additional high-affinity site ( K d = 0.4 nM), as well as the low-affinity site, was found in adult tissue. Similar studies were carried out with cultures of cerebral neurons prepared from 8-day embryos. The level of specific binding of muscimol, Flu and TBPS increased in culture. achieved one half of the maximum level by days 4–5, maximal levels by day 10 and decreased slowly thereafter. The maximal levels in culture corresponded, respectively, to 27%, 67% and 57% of these found in the 18-day embryo. The binding of Flu to membranes from neurons, embryos and adults was enhanced by addition of GABA while TBPS binding was inhibited. The EC 50 and IC 50 values for these effects corresponded to those for gating of chloride channels. These findings indicate a coordinated expression of receptors for GABA, benzodiazepines and convulsant/TBPS during neuronal maturation both in vivo and in vitro. The schedule for this postsynaptic ontogeny is very similar to that for presynaptic markers of GABAergic neurons (see companion paper).

Benzodiazepine anticonvulsants accelerate and β-carboline convulsants decelerate the kinetics of [ 35S]TBPS binding at the chloride ionophore

The effect of flunitrazepam and DMCM was examined on the kinetics of [ 35S]t-butylbicyclophosphorothionate (TBPS) binding. The enhancing effect of flunitrazepam and the decreasing effect of DMCM on TBPS binding was due o non-equilibrium conditions. The anticonvulsant benzodiazepine increased both the association and dissociation rate constants. The convulsant β-carboline had the opposite effect. Benzodiazepine receptor ligands might affect the convulsant TBPS sites parallel with opening/closing of the chloride ionophores.

Multiple [35S]t-butylbicyclophosphorothionate binding sites in rat and chicken cerebral hemispheres.

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to membranes from cerebral hemispheres of adult rat and chicken was determined over a range of radioligand concentrations from 0.25 to 500 nM. Scatchard plots of these data were curvilinear and nonlinear regression analysis indicated binding to two sites that differ in affinity. For rat cerebrum, KD(1) = 1.15 nM, Bmax(1) = 0.085 pmol/mg; KD(2) = 232 nM, Bmax(2) = 16.9 pmol/mg. For chicken cerebrum, KD(1) = 1.39 nM, Bmax(1) = 0.111 pmol/mg; KD(2) = 166 nM, Bmax(2) = 17.6 pmol/mg. This multiplicity of [35S]TBPS binding was further confirmed when unlabeled TBPS or picrotoxinin displaced radioligand. The displacement curves were biphasic and yielded Hill coefficients from 0.65 to 0.70. These displacement curves were also resolved into two components with distinct IC50 values for unlabeled TBPS (rat, 1.55 and 271 nM; chicken, 2.40 and 224 nM). The IC50 values were similar to the dissociation constants obtained from equilibrium binding measurements.

Gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]- t-butylbicyclophosphorothionate binding to cerebellar granule cells

t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, we have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [35S]TBPS. At 25 degrees C, and in a modified Locke solution, the [35S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [35S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [35S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [35S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [35S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [35S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of in vitro and in vivo ethanol administration on [ 35S]t-Butylbicyclophosphorothionate binding in C57 mice

Previous studies have shown that ethanol may produce some of its effects by facilitation of GABAergic transmission. One of the potential sites of drug action at the GABA receptor complex is the picrotoxin site, which can be studied with [ 35S]t-butylbicyclophosphorothionate (TBPS). Ethanol inhibited the binding of [ 35S]TBPS to C57 mice brain regions in vitro. This inhibition appears to be noncompetitive since ethanol decreased the B max and not the K D value of [ 35S]TBPS. C57 mice were chronically treated with ethanol in liquid diet to determine if the sensitivity of TBPS binding is altered following chronic treatment or during withdrawal. Chronic treatment with ethanol and during withdrawal did not alter the K D or B max values of [ 35S]TBS binding in C57 mice brain regions. It is suggested that the sensitivity of picrotoxin site on the oligomeric GABA receptor complex is not altered during ethanol tolerance or withdrawal. The effects of ethanol on GABA system may be mediated by its interaction with the coupling mechanism(s) or a direct effect on the chloride channels.

Binding of GABA receptor channel drugs to a putative voltage-dependent chloride channel in torpedo electric organ

In muscle and neuronal cells, there are two major types of Cl − channels: the voltage-dependent Cl − channels, which are activated by changes in voltage across the membrane and contribute a major share to the resting membrane conductance (1), and the chemically operated Cl − channels which are parts of the inhibitory receptor systems of γ-aminobutyric acid (GABA) or glycine, and are activated by binding of the transmitter (2). Although much is known about the biochemical and pharmacological nature of these receptors, very little such information is available on the voltage-dependent Cl − channels. The opportunity to study one such channel became feasible when it was discovered that the electric organ of electric rays, which is a rich source for nicotinic acetylcholine (ACh) receptors, may also be rich in Cl − channels. An anion-selective channel was discovered in the electric organs of Torpedo californica as shown by increased anion conductance in reconstituted membrane vesicles in planar phospholipid bilayer (3) and in liposomal membrane vesicles (4), and of Narke japonica as shown by changes in osmotic volume and efflux of 35SO 4 − (5). These effects were inhibited by the specific Cl − channel inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). This Cl − channel was also expressed in frog Xenopus oocytes that had been injected with mRNA fraction from Torpedo electric organ, while another fraction of mRNA induced the incorporation of ACh receptors (6). This electric organ Cl − channel is highly anion selective (at least 20-fold more permeable to Cl − than to K +) compared to a 2-fold selectivity for a mitochondrial Cl − channel (3). The present study was initiated to attempt identification of the Cl − channel in Torpedo electric organ, using [ 35 S]t- butylbicyclophosphorothionate (TBPS) as a probe since it binds with a high affinity to the GABA receptor Cl − channel (7), and to determine and compare the drug specificities of these different types of Cl − channels.

Separate site(s) of action of optical isomers of 1-methyl-5-phenyl-5-propylbarbituric acid with opposite pharmacological activities at the GABA receptor complex

The behavioral profile of the optical isomers of 1-methyl-5-phenyl-5-propylbarbituric acid (MPPB) and their interaction with the convulsant binding site at the GABA receptor complex were investigated. R(−)-MPPB produced dose-related loss of righting reflex, whereas S(+)MPPB produced convulsions in a dose-dependent manner. Subconvulsive doses of S(+)MPPB were proconvulsant with a subeffective dose of picrotoxin. S(+)MPPB-induced seizures were blocked by R(−)MPPB and pentobarbital. In contrast, S(+)MPPB did not block the loss of righting reflex produced by R(−)MPPB or pentobarbital. S(+)MPPB inhibited the binding of [ 35S]t-butylbicyclophosphorothionate (TBPS), a ligand that binds to the picrotoxin site on the oligomeric GABA receptor complex, to rat brain membranes competitively, whereas R(−)MPPB inhibited it noncompetitively. Thus, the optical isomer of MPPB, which have opposite pharmacological effects, interact differently with the convulsant (TBPS) site at the GABA receptor complex. These results suggest that the convulsant S(+)MPPB and the depressant R(−)MPPB may produce their behavioral effects by acting via convulsant and anticonvulsant sites, respectively, at the GABA receptor complex.

Avermectin B1a modulation of gamma-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.

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.

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<0.001) between their relative affinities for the [ 35S]-TBPS binding site and their convulsant potencies, indicating that pentamethylenetetrazol and related tetrazoles may produced their convulsant and anxiogenic actions via the GABA-benzodiazepine-chloride ionophore receptor complex.

Differential interactions of GABA agonists, depressant and convulsant drugs with [ 35S]-t-butylbicyclophosphorothionate binding sites in cortex and cerebellum

Effects of three GABA agonists, four GABA antagonists and convulsants (picrotoxinin, α-dihydropicrotoxinin [DHP], pentamethylenetetrazole [PTZ] and isopropylbicyclophosphate ester) and three depressant drugs (pentobarbital (+)etomidate and etazolate) were investigated on [ 35S]t-butylbicyclophosphorothionate (TBPT) in cortex and cerebellum. All the convulsants tested were equipotent in inhibiting [ 35S]TBPT binding in cortex and cerebellum. Convulsants like picrotoxinin inhibited [ 35S]-TBPT binding competitively in both cortex and cerebellum. In contrast, γ-aminobutyric acid (GABA) agonists (muscimol, GABA and 4,5,6,7-tetrahydroisoxazol[5,4- C]pyridine-3-ol[THIP]), and depressants like etazolate, (+)etomidate ande more potent inhibitors of [ 35S]TBPT binding in cerebellum than in cortex. GABA inhibition of [ 35S]TBPT binding appears to be mediated through a low-affinity site. GABA and pentobarbital inhibited [ 35S]TBPT binding in cortex and cerebellum noncompetitively. Depressants like pentobarbital appear to interact with the TBPT sites allosterically. These results suggest that depressant and convulsant drugs that modulate GABAergic transmission interact differently with the TBPT binding sites in cortex and cerebellum.

Interactions of lindane, toxaphene and cyclodienes with brain-specific [formula omitted]-butylbicyclophosphorothionate receptor

Three major classes of chlorinated hydrocarbon insecticides, i . e ., the lindane/hexachlorocyclohexane, toxaphene and aldrin/dieldrin types, are potent, competitive, and stereospecific inhibitors of t -butylbicyclophosphorothionate (TBPS) binding to brain-specific sites, thereby indicating an action at the γ-aminobutyric acid (GABA)-regulated chloride channel. The most inhibitory and toxic of four isomers of hexachlorocyclohexane is lindane and of >188 components of toxaphene is 2,2,5- endo , 6- exo , 8,9,9,10-octachlorobornane. 12-Ketoendrin (IC 50 = 36 nM) is twice as active as the most potent previously known inhibitor of TBPS binding and it is also the most inhibitory and toxic of 22 cyclodienes examined. Within each of these three series of polychlorocycloalkanes the mammalian toxicity is closely related to the potency for inhibition of TBPS binding. A modified receptor assay incorporating liver microsomes and reduced nicotinamide-adenine dinucleotide phosphate compensates in part for oxidative detoxification and bioactivation. Specific TBPS binding is reduced in a dose-dependent manner in dieldrin-poisoned rats. DDT, mirex and kepone are not inhibitors of TBPS binding, even at 10 μM.

Purification of the GABA/benzodiazepine/barbiturate receptor complex

The post synaptic receptor for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is a complex protein containing a chloride channel and modulatory sites for two classes of drugs, the benzodiazepines (BZ) and convulsants/barbiturates. Barbiturates which enhance GABA-activated chloride permeability at the tissue and cellular level also are able to enhance benzodiazepine and GABA receptor binding at the molecular level. Convulsant/barbiturate sites can be assayed directly with [ 35S]t-butyl bicyclophosphorothionate (TBPS). This activity was solubilized with the detergent CHAPS and shown to co-migrate with BZ and GABA receptor binding activity during protein fractionation as a single protein complex, which could be purified intact by over 1000-fold on benzodiazepine affinity columns.

A gamma-aminobutyric acid/benzodiazepine receptor complex from bovine cerebral cortex. Improved purification with preservation of regulatory sites and their interactions.

A gamma-aminobutyrate/benzodiazepine receptor complex has been purified from bovine cerebral cortex by an improved procedure using a zwitterionic detergent. A high affinity binding site for the chloride ion channel-blocking ligand [35S]t-butyl bicyclophosphorothionate ( TBPS ) was co-purified with the high affinity binding sites for gamma-aminobutyrate and benzodiazepines. The latter two have previously been shown to reside on the same physical structure ( Sigel , E., Stephenson , F.A., Mamalaki , C., and Barnard , E. A. (1983) J. Biol. Chem. 258, 6965-6971). The dissociation constants, as measured in assay medium containing zwitterionic detergent were 90 +/- 20 nM for TBPS and 11 +/- 4 nM for [3H]flunitrazepam, whereas the binding of [3H]muscimol, a gamma-aminobutyrate agonist, showed a more complex binding behavior with more than one site. If the same preparation was assayed in a medium containing instead Triton X-100 as the detergent, the binding of TBPS was strongly inhibited, [3H]flunitrazepam binding was unaffected, and [3H]muscimol bound to a single class of sites with a dissociation constant of 33 +/- 3 nM. Regulatory interactions were retained in the complex isolated by the improved method: [3H]flunitrazepam binding was stimulated by gamma-aminobutyrate or by pentobarbital, and in a dose-dependent manner. The same two subunit types of Mr = 53,000 and 57,000 are present in the purified receptor complex as previously reported.

35S]-t-butylbicyclophosphorothionate binding sites are constituents of the gamma-aminobutyric acid benzodiazepine receptor complex

t- Butylbicyclophosphorothionate ( TBPS ), a derivative of potent GABA antagonistic cage convulsants, has recently been introduced ( Squires , R. F., J.E. Casida , M. Richardson, and E. Saederup (1983) Mol. Pharmacol. 13:326-336) as ligand for a GABA-A receptor-linked drug receptor. Using conventionally prepared washed membrane fractions from rat cerebral cortex, we have confirmed that in the presence of 200 mM NaBr [35S] TBPS binds to a high affinity population of binding sites (Kd 26 +/- 5 nM) and that muscimol inhibits [35S] TBPS binding (IC50 0.32 microM) allosterically. In 200 mM NaCl the apparent affinity of [35S] TBPS binding sites is lower (Kd 60 +/- 5 nM), and muscimol has biphasic effects with stimulation at low concentrations of muscimol (EC50 0.023 microM) followed by inhibition at high concentrations (IC50 0.72 microM). Both base line [35S] TBPS binding (in 200 mM NaCl) and muscimol inhibition of [35S] TBPS binding (in 200 mM NaBr) are bidirectionally modulated by the occupancy of benzodiazepine receptors with its ligands. Benzodiazepine receptor agonists, regardless of their structure, enhance and inverse benzodiazepine receptor agonists inhibit base line [35S] TBPS binding and muscimol inhibition of [35S] TBPS binding. Fourteen ligands for benzodiazepine receptors display a similar in vitro profile as benzodiazepine receptor agonists or inverse benzodiazepine receptor agonists on [35S] TBPS binding as their anti- or proconvulsive effects in vivo suggest (Jensen, L. H., E. N. Petersen, and C. Braestrup (1983) Life Sci. 33: 393-399). That [35S] TBPS binding sites are constituents of a GABA benzodiazepine receptor complex is also suggested by a number of membrane pretreatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of pentamethylenetetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex.

The interactions of pentamethylenetetrazole and ten tetrazole analogues with the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex was investigated. All the active tetrazole analogues potently inhibited the binding of [35S]t-butyl- bicyclophosphorothionate ( TBPT ), a ligand which binds to the picrotoxinin site. Tetrazole analogues appear to interact with the picrotoxinin site competitively. All the tetrazole analogues tested were more potent in inhibiting TBPT binding than diazepam binding. There is a reasonably good correlation between the tetrazole analogues to inhibit TBPT binding and the doses at which they produce convulsions. Pentamethylenetetrazole inhibited TBPT binding at concentrations which are similar to the in vivo concentrations present during convulsions produced by this drug. These results suggest that tetrazoles may produce convulsions by acting at the picrotoxin-sensitive site of the benzodiazepine-GABA receptor-ionophore complex.

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 IC 50 value of ∼ 20 μM. 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.

Binding characteristics and interactions of depressant drugs with [35S]t-butylbicyclophosphorothionate, a ligand that binds to the picrotoxinin site.

[35S]t-Butylbicyclophosphorothionate (TBPT), a cage convulsant with picrotoxinin-like activity, binds to rat brain membranes to a single site with an apparent KD of 25.1 +/- 5.6 nM and a Bmax of 1.40 +/- 0.22 pmol/mg protein. TBPT binding to rat brain membranes was inhibited by a variety of convulsant, depressant, anxiolytic, and anticonvulsant drugs that had previously been shown to inhibit [3H]alpha-dihydropicrotoxinin binding. Depressant drugs such as pentobarbital and the nonbarbiturate (+)etomidate inhibited TBPT binding in an uncompetitive manner. Thus, pentobarbital and (+)etomidate decreased both the affinity and the number of binding sites of TBPT to whole brain membranes. The IC50 values of (+)etomidate (9 microM) and pentobarbital (90 microM) are similar to the EC50 values at which they enhance both [3H]gamma-aminobutyric acid and [3H]diazepam binding in cerebral cortex membranes. RO5-4864, which has recently been shown to be a convulsant, also inhibited TBPT binding (IC50 = 10 microM). These results suggest that TBPT binds to the picrotoxinin site and further supports the notion that the picrotoxinin site is an important modulatory site at the benzodiazepine-GABA receptor-ionophore complex.

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

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 GABAA agonist [3H]muscimol and for the chloride ion channel blocking agent [35S]t-butylbicyclophosphorothionate (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 53 000 and Mr 57 000. 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 53 000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48 000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires greater than 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments less than Mr 10 000.

Chemical Nature of Synaptic Transmission in Vertebrates

Chemical Nature of Synaptic Transmission in Vertebrates