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.     

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.     

Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands

gamma-aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha5 subunit were substituted by their corresponding alpha1 residues. Given the high affinity and selectivity of alpha1-containing compared with alpha5-containing GABAA receptors for zolpidem, mutated alpha5 subunits were co-expressed with beta2 and gamma2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha5P162T and alpha5E200G, might alter binding pocket conformation, whereas alpha5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha1 subunit, particularly alpha1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha1-containing receptors, and a model of this binding pocket is presented.     

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [³H]FLU and [³H]propyl beta-carboline-3-carboxylate ([³H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [³H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10^?4 M) on the $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition curve was detected. In contrast, when assays were carried out at 0°C using [³H]FLU or a high concentration of [³H]PCC to achieve [³H]ligand receptor occupancy of both type I and type II receptors, GABA (10^?4 M) caused a significant increase in the affinity of FLU as measured by $\text{FLU}\text{[}{}^3\text{H]}\text{FLU}$ and $\text{FLU}\text{[}{}^3\text{H]}\text{PCC}$ competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the $\text{PCC}\text{[}{}^3\text{H]}\text{FLU}$ competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.     

Biochemical pharmacology of the gamma-aminobutyric acid receptor/ionophore protein

The synaptic receptor sites for the neurotransmitter gamma-aminobutyric acid (GABA) can be assayed in vitro with several radiolabeled agonists and one antagonist. Numerous criteria of specificity have been met for these binding sites. All of the ligands show heterogeneity in binding affinities. The subpopulations thus defined have a remarkably similar specificity for GABA analogs, which suggests an intimate relationship and possible interconvertibility. Modulation of GABA receptor binding by barbiturates, anions, and other membrane treatments that affect agonists and antagonists in an opposite manner suggests a three-state model of interconvertible affinities. The complex of GABA receptor and chloride ion channel contains modulatory sites for barbiturates and benzodiazepines, drugs that enhance GABA responses in neurons. The receptor complex can be solubilized in detergent with the three mutually interacting receptor activities intact. The complex has an apparent molecular weight of 355,000 and has been partially purified. GABA agonist function has been assayed at the biochemical level by measuring the activation of 36Cl- efflux from preloaded hippocampal slices by GABA, muscimol, and barbiturates. This response is blocked by the antagonists of the GABA site (bicuculline) and the barbiturate site (picrotoxin). Comparison of binding and function on the same tissue should be useful in analyzing the mechanism of action of GABA.     

Multiple benzodiazepine receptors and their regulation by gamma-aminobutyric acid

The interaction of propyl β-carboline-3-carboxylate (PCC) with benzodiazepine receptors in the cerebral cortex of the rat was investigated by direct measurements of [³H]PCC binding and by competitive inhibition of [³H]flunitrazepam (FLU) binding. Initial experiments showed that [³H]PCC binding exhibited characteristics of saturability, stereospecificity and a pharmacological specificity remarkably similar to that of [³H]FLU binding. Analysis of [³H]PCC binding isotherms and PCC/[³H]PCC competition curves revealed the presence of a small population of super high affinity PCC binding sites (K_SH = 30–100 pM) which represents approximately 3–6% of the total sites. When measured by competitive inhibition of [³H]FLU binding, receptor occupancy by PCC was generally consistent with that determined by direct measurements of [³H]PCC binding. Analysis of the PCC/[³H]FLU competition curve revealed the presence of two major populations of high and low affinity PCC binding sites with dissociation constants of 0.54 and 10 nM and relative abundances of 52 and 45%, respectively. Collectively, the results of the [³H]PCC binding isotherm, PCC/[³H]PCC competition curve and PCC/[³H]FLU competition curve are internally consistent when rationalized in terms of three populations of benzodiazepine receptors - super high, high, and low affinity - each having different affinities for PCC and equal affinity for FLU. The effects of γ-aminobutyric acid (GABA) on PCC and FLU binding were investigated, and it was observed that GABA enhanced the binding of FLU to the various receptor subtypes whereas no significant effect of GABA on the binding of PCC was detected.     

The type I and type II gamma-aminobutyric acid/benzodiazepine receptor: 1. Purification and two-dimensional electrophoretic analysis of the receptor from cortex and cerebellum

The gamma-aminobutyric acid/benzodiazepine receptor complex was purified from rat cortex and cerebellum by benzodiazepine affinity chromatography. Receptors purified from cortex and cerebellum showed different relative affinities for Cl 218872, a non-benzodiazepine ligand which discriminates type I and type II receptors. In contrast, no differences in subunit composition could be detected between these two purified receptor preparations when analyzed by two-dimensional gel electrophoresis.     

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.     

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.     

The gamma-aminobutyric acid receptor in catfish brain

When the binding of [3H]gamma-aminobutyric acid (GABA) to its receptor in catfish synaptic membranes was studied, a high affinity (Kd = 8.4 nM) and a low affinity (Kd = 65 nM) binding component was observed. Muscimol, thiomuscimol, tetrahydroisoxazole-5,4-c-pyridin-3-ol, imidazole acetic acid and bicuculline each competitively inhibited both high affinity and low affinity [3H]GABA binding. The potency of these inhibitors was similar to that reported for the GABA receptor from mammalian brain. It is concluded that the GABA receptor from catfish brain has very similar properties to the receptor from mammalian central nervous system and consequently has not undergone any obvious evolutionary changes.     

Stimulation of benzodiazepine binding to rat brain membranes and solubilized receptor complex by avermectin B1a and gamma-aminobutyric acid

The binding of [3H]flunitrazepam to benzodiazepine receptors in synaptic membranes and a digitonin-solubilized receptor fraction of rat brain is increased by avermectin B1a and gamma-aminobutyric acid (GABA). The effects of avermectin B1a and GABA are both sensitive to inhibition by (+)-bicuculline. Avermectin B1a and GABA both decrease the Kd and increase the Bmax of [3H]flunitrazepam binding to membranes. Kinetic analysis of the binding of [3H]flunitrazepam to rat brain membranes indicates that avermectin B1a and GABA reduce the rate constants of both association and dissociation between the ligand and the receptor. These results suggest a similar mechanism of modulation of benzodiazepine binding by avermectin B1a and GABA. This modulation may involve in interaction among the receptors for benzodiazepine, GABA and avermectin B1a.     

Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding

GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.     

Function of gamma-aminobutyric acid receptor/channel rho 1 subunits in spinal cord

gamma-Aminobutyric acid (GABA) receptor/channel rho 1 subunits are important components in inhibitory pathways in the central nervous system. However, the precise locations and roles of these receptors in the central nervous system are unknown. We studied the expression localization of GABA receptor/channel rho 1 subunit in mouse spinal cord and dorsal root ganglia (DRG). The immunohistochemistry results indicated that GABA receptor/channel rho 1 subunits were expressed in mouse spinal cord superficial dorsal horn (lamina I and lamina II) and in DRG. To understand the functions of the GABA receptor/channel rho 1 subunit in these crucial sites of sensory transmission in vivo, we generated GABA receptor/channel rho 1 subunit mutant mice (rho 1-/-). GABA receptor/channel rho 1 subunit expression in the rho 1-/- mice was eliminated completely, whereas the gross neuroanatomical structures of the rho 1-/- mice spinal cord and DRG were unchanged. Electrophysiological recording showed that GABA-mediated spinal cord response was altered in the rho 1-/- mice. A decreased threshold for mechanical pain in the rho 1-/- mice compared with control mice was observed with the von Frey filament test. These findings indicate that the GABA receptor/channel rho 1 subunit plays an important role in modulating spinal cord pain transmission functions in vivo.     

Channel opening of gamma-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses

The function of gamma-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36Cl- isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36Cl- influx was completed in three phases of ca. 3% (t 1/2 = 0.6 s), 56% (t 1/2 = 82 s), and 41% (t 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36Cl- influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles with initial first-order rate constants of 9.5 s-1 and 2.3 s-1 and desensitizing with first-order rate constants of 21 s-1 and 1.4 s-1, respectively, at saturation. The half-response concentrations were similar for both receptors, 150 microM and 114 microM GABA for desensitization and 105 microM and 82 microM for chloride exchange, for the faster and slower desensitizing receptors, respectively. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of "low-affinity" to "high-affinity" GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The benzodiazepine agonist clonazepam potentiates the effects of gamma-aminobutyric acid on alpha-MSH release from neurointermediate lobes in vitro

The action of the central-type benzodiazepine-receptor agonist clonazepam on alpha-MSH release has been studied in vitro using perifused frog neurointermediate lobes. High concentrations of clonazepam (3.16 X 10(-5) and 10(-4) M) caused an inhibition of alpha-MSH release and this effect was reversed by the central-type benzodiazepine-receptor antagonist Ro 15-1788. High doses of GABA (10(-5) and 10(-4) M) induced a biphasic effect on pars intermedia cells: a brief stimulation followed by a sustained inhibition of alpha-MSH secretion. Administration of clonazepam (10(-5) M) in the presence of various concentrations of GABA (10(-6) to 10(-4) M) led to a potentiation of both stimulatory and inhibitory phases of alpha-MSH secretion induced by GABA. Ro 15-1788 completely abolished the potentiating effect of clonazepam. Thus our results indicate that endogenous benzodiazepine receptors may modulate the effects of GABA on alpha-MSH secretion.     

Mapping the benzodiazepine photoaffinity-labelling site with sequence-specific gamma-aminobutyric acidA-receptor antibodies.

The gamma-aminobutyric acidA (GABAA) receptor purified from adult bovine cerebral cortex was photoaffinity-labelled with the agonist benzodiazepine [3H]flunitrazepam and the radioactivity shown to be coincident with a band with Mr 53,000 that was recognized by three anti-(GABAA receptor alpha 1 subunit sequence)-specific antibodies. Complete and limited CNBr cleavage of the purified photoaffinity-labelled receptor was carried out. The products of this reaction were analysed for radioactivity, for immunoreactivity with anti-[alpha 1-(1-15)-peptide], anti-[alpha 1-(324-341)-peptide] and anti-[alpha 1-(413-429)-peptide] polyclonal antibodies and for carbohydrate by biotinylated concanavalin A lectin overlay. Complete CNBr cleavage gave a radioactive peptide with Mr 10,000-12,000 that was not recognized by the above-mentioned specific antisera. By using the deduced amino acid sequence of the alpha 1 subunit [Schofield, Darlison, Fujita, Burt, Stephenson, Rodriguez, Rhee, Ramachandran, Reale, Glencorse, Seeburg & Barnard (1987) Nature (London) 328, 221-227], it is proposed that the site of the benzodiazepine-agonist photoaffinity-labelling reaction does not lie within the amino acid sequences alpha 1 1-58 and alpha 1 149-429.