Pharmacological and Biochemical Properties of the γ-Aminobutyric Acid–Benzodiazepine Receptor Protein from Codfish Brain

The gamma-aminobutyric acidA (GABAA) receptor of codfish brain has been purified to homogeneity and contains a single polypeptide band of 56 kDa molecular mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of codfish GABA receptor photoaffinity-labeled by both [3H]flunitrazepam ([3H]Flu) and [3H]muscimol showed a single radioactive peak with molecular mass of 56 kDa, in contrast to the multiple subunits found in other vertebrate species. The codfish receptor, purified using benzodiazepine (BZ, Ro 7-1986/1) affinity chromatography, contains an apparent single band both by isoelectric focussing and on a silver-stained SDS gel. The receptor density and affinity constants for [3H]muscimol and [3H]Flu binding are comparable to those in mammalian brain, and the specific activity (greater than 1,000 pmol/mg of protein) is comparable to that of preparations purified from those sources. The pharmacological specificity of the codfish GABA-BZ receptor is generally similar to that of mammalian brain, including GABA-BZ coupling. The BZ binding exhibits homogeneous kinetic properties resembling those of the mammalian BZ2 receptor type, and shows strong GABA enhancement of [3H]Flu binding and weaker pentobarbital potentiation. This is consistent with other observations of an earlier phylogenetic, as well as ontogenetic, emergence in mammals of the BZ2 receptor subtype than the BZ1. Codfish GABA receptor is postulated to be a homo-oligomer in which the conformation of GABA and BZ recognition sites is very similar to that in the mammalian hetero-oligomeric GABAA receptor. The codfish receptor appears to be encoded by an ancestral gene and indicates an early development of BZ-GABA coupling.     

A single histidine in GABAA receptors is essential for benzodiazepine agonist binding.

Benzodiazepines (BZ) modulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A (GABAA) receptor, the major inhibitory ion channel in the mammalian brain. This receptor is composed of structurally distinct subunits whose numerous molecular variants underlie the observed diversity in the properties of the BZ site. Pharmacologically distinct BZ sites can be recreated by the recombinant coexpression of any one of six alpha subunits, a beta subunit variant, and the gamma 2 subunit. In these receptors the alpha variant determines the affinity for ligand binding of the BZ site. Notably, the alpha 1 and alpha 6 variants impart on alpha chi beta 2 gamma 2 receptors high and negligible affinity, respectively, to BZ ligands with sedative as well as anxiolytic activities. By exchanging domains between the alpha 1 and alpha 6 variants, we show that a portion of the large extracellular domain determines sensitivity toward these ligands. Furthermore, we identify a single histidine residue in the alpha 1 variant, replaced by an arginine in alpha 6, as a major determinant for high affinity binding of BZ agonists. This residue also plays a role in determining high affinity binding for BZ antagonists. Hence, this histidine present in the alpha 1, alpha 2, alpha 3, and alpha 5 subunits appears to be a key residue for the action of clinically used BZ ligands.     

Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex

The autoradiographic localization of subcomponents of the gamma-aminobutyric acid (GABA) receptor-chloride ionophore complex has provided insight into the distribution of this macromolecular system. GABA inhibits neurons by preferentially increasing the permeability of the affected membrane to chloride ions. This inhibition can be modified by the presence of other substances which bind to the GABA receptor complex. Autoradiographic localization of specific receptor subtypes associated with this complex has been accomplished in the central nervous system. This type of analysis has been performed on high and low affinity GABAA, benzodiazepine (BZ; both BZ1 and BZ2) and convulsant sites. These receptor sites are situated in distinct brain regions and co-exist in several areas. Other receptor subtypes, which may be influenced by the presence of GABA, can be analyzed for comparison in order to define regions of the brain where GABA may be exerting independent effects (i.e., those not associated with chloride channels). Microscopic localization of receptor sites indicates specific areas to investigate in further studies concerning the characterization of subcomponents of the macromolecular GABA complex associated with chloride ion channels.     

Differential expression of GABAA receptor alpha-subunits in rat brain during development.

Unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor have been expressed in E. coli and used to generate polyclonal antisera specific for these subunits. The antibodies identify proteins by SDS-polyacrylamide gel electrophoresis and western blotting of molecular size 51 kDa, 53 kDa, 59 kDa and 55 kDa, respectively, which show differential patterns of expression during development. Whereas the alpha 2 and alpha 3 subunits are present at early stages, the expression of alpha 1 and alpha 3 subunits is low at birth and increases with age. This differential expression could be correlated with previous studies examining the developmental expression of BZ1 and BZ2 benzodiazepine 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)     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [3H]Ro 15–4513: A Biochemical and Autoradiographic Study

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.     

Relationship of GABAa receptor heterogeneity to regional differences in drug response

Molecular biological approaches to the GABAa receptor have resulted in new insights into the structure and pharmacology of this complex. It is known that the GABAa complex is a heterooligomer composed of multiple subunits which contain binding sites for the GABA, benzodiazepines and barbiturates. These subunits also contain regulatory sites for phosphorylation by intracellular kinases. There appear to be regional differences in the expression of the various subunits for the GABAa receptor complex. The functional significance of molecular heterogeneity is not yet known but it is expected that regional differences may result in pharmacologically diverse responses. Studies on the effects of chronic administration of diazepam have clearly delineated such regional differences. Chronic benzodiazepine administration results in the development of subsensitivity to the electrophysiological actions of GABA in the dorsal raphe, but not in GABA receptive neurons of the substantia nigra pars reticulata. Such data is consistent with regional heterogeneity in response to chronic benzodiazepine, exposure. It is hoped that by understanding GABAa receptor heterogeneity, and its molecular basis, we can improve the, existing receptor subtype specificity and pharmacology of the benzodiazepines.     

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

Abstract: The chemical topography of the γ-aminobutyric acid (GABA) and benzodiazepine (BZ) receptors was investigated in a thoroughly washed cortical membrane preparation of the rat. Chemical modification by several amino- and tyrosyl-selective reagents and the protection from it by direct and allosteric ligands of the GABA-BZ receptor complex were used to identify the residues at the binding sites. Inhibition of specific GABA binding by p -diazobenzenesulfonic acid (DSA), tetrani-tromethane (TNM), and N -acetylimidazole and the selective and complete protection from it by GABA and muscimol suggest the presence of a tyrosine residue at the GABA_A site. TNM, like DSA, selectively decreased the number of the low-affinity GABA receptors, and this could be completely protected only by GABA concentrations that can saturate the low-affinity sites. TNM pre-treatment also abolished the muscimol enhancement of [³H]diazepam binding, which suggests that the low-affinity GABA receptor sites are responsible for this enhancement. Inhibition of GABA binding by pyridoxal-5-phosphate (PLP) and the selective protection by GABA and muscimol support the presence of a lysine residue at the GABA_A receptor site. Complete and selective protection from diethylpyrocarbonate (DEP) inhibition of [³H]diazepam binding by flurazepam suggests the presence of a histidine residue at the BZ site. Flurazepam selectively protected from inhibition of [³H]diazepam binding by N -bromosuccinimide and N -acetylimidazole, but not that by DSA and TNM, which does not allow a unanimous conclusion regarding the presence of tyrosine or tryptophan residues at the BZ site.     

Modulatory Effects of Thyroxine Treatment on Central and Peripheral Benzodiazepine Receptors in the Rat

The effects of 10 days of D-thyroxine (T4) treatment on central benzodiazepine (BZ) receptors in the brain and on peripheral-type BZ binding sites in the heart, kidney, and testis of rats were studied. The experimental hyperthyroidism resulted in an increase in the density of cortical central BZ receptors, without any alteration of the affinity of the receptors to [3H]flunitrazepam. The increase in cortical central BZ receptors was also accompanied by the up-regulation of peripheral BZ binding sites in the heart, kidney, and testis. The affinity of the peripheral BZ binding sites for the ligand [3H]PK 11195 was not affected by T4 treatment in any of these three organs. The increase in the density of brain cortical central BZ receptors was less prominent than the increase in the peripheral BZ binding sites. The modulatory effect of T4 treatment on central and peripheral BZ receptors might be attributed to the direct interaction of the thyroid hormone at these sites or might reflect a physiological compensatory adaptation mechanism to thyrotoxicosis associated with hypermetabolism, anxiety, and stress.     

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.     

Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and short-sleep mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of 3H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.     

Binding of chlorinated benzidines to the rat hepatic aromatic hydrocarbon receptor

The binding of benzidine, 3,3'-dichlorobenzidine (3,3'-Cl2BZ), and the asymmetrically-substituted chlorinated benzidines 3,5-dichlorobenzidine (3,5-Cl2BZ) and 3,5,3'-trichlorobenzidine (Cl3BZ) to the rat hepatic cytosolic aromatic hydrocarbon (Ah) receptor was measured, in order to assess the mechanism of P-450I induction by 3,3'-Cl2BZ. Cl3BZ is the most mutagenic benzidine derivative in the Ames assay. Binding affinity to the Ah receptor protein was determined by displacement of labelled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the receptor, measured with the sucrose density gradient centrifugation technique. The rank order of affinities and the apparent inhibitor constants were: Cl3BZ (4 microM) greater than 3,5-Cl2BZ (8.4 microM) greater than 3,3'-Cl2BZ (10 microM). Benzidine did not displace TCDD from the receptor protein. 4-Aminobiphenyl a structural link between the benzidine and biphenyl series competed weakly with TCDD. The 50% inhibition concentration was about 150 microM. The results are consistent with the hypothesis that the induction of P-450 enzymes by 3,3'-Cl2BZ in vivo is mediated by the Ah receptor.     

Characterization of six partial deletions in the low-density-lipoprotein (LDL) receptor gene causing familial hypercholesterolemia (FH).

Two hundred thirty-four unrelated heterozygotes for familial hypercholesterolemia (FH) were screened to detect major rearrangements in the low-density-lipoprotein (LDL) receptor gene. Total genomic DNA was analyzed by Southern blot hybridization to probes encompassing exons 1-18 of the LDL receptor gene. Six different mutations were detected and characterized by the use of exon-specific probes and detailed restriction mapping. Each mutation is unique and suggests that molecular heterogeneity underlies the molecular pathology of FH. There appear to be preferential sites within the LDL receptor gene for major rearrangements resulting in deletions.     

Discovery of novel insomnia leads from screening traditional Chinese medicine database

Insomnia is a prominent modern disease that affects an increasing population. Undesirable side effects of commercial drugs highlight the need to develop novel insomnia drugs. Virtual screening of traditional chinese medicine Database@Taiwan (TCM Database@Taiwan) identified 2-O-Caffeoyl tartaric acid (1), 2-O-Feruloyl tartaric acid (2), and Mumefural (3) as potential agonists for both gamma-amino butyric acid (GABA) or benzodiazepine (BZ) binding sites. The TCM candidates exhibited higher affinity than GABA and Zolpidem, a phenomenon that could be attributed to higher quantity of stabilizing H-bonds. Efficacy profiles using support vector machines and pharmacophore contour also suggest drug potential of the TCM candidates. Fragments added to the de novo derivatives 3a, 3b, 3c for GABA binding site, and 1a, 2a, and 3d for BZ binding site contributed to new binding sites and structural stability, further optimizing binding to GABA or BZ binding sites. Increased opening of the ion channel by candidate ligands provide strong support for their potential biological functions. The dual binding properties of the TCM candidates present a unique opportunity to develop twin-targeting drugs with less side effects. Derivative structures can be used as starting points for developing high affinity GABA_A receptor agonists with specificity towards GABA binding site and BZ binding site.     

Sequence and Regional Distribution of the mRNA Encoding the α2 Polypeptide of Rat γ-Aminobutyric AcidA Receptors

A cDNA from a rat hippocampal cDNA library encodes an isoform of the alpha polypeptide of the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the alpha 2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor alpha polypeptides and shares 71-76% identity with previously described rat alpha polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the alpha 2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined "BZ type II" receptors. Other workers have previously shown that the alpha polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of alpha 2 mRNAs in rat brain suggests that the alpha 2 subunit may indeed be involved in the BZ type II receptors.     

Beta-tubulin complementary DNA sequence variations observed between cyathostomins from benzimidazole-susceptible and -resistant populations

The molecular mechanism of benzimidazole (BZ) resistance in cyathostomins of horses is still unclear. Previous studies revealed that the TTC or TAC polymorphism in codon 200 of the beta-tubulin isotype 1 gene is not as strictly correlated with BZ resistance as in trichostrongyles in sheep. To identify further sites of polymorphism within the beta-tubulin gene related to BZ resistance, complete complementary DNAs (cDNAs) encoding beta-tubulin of adult worms of Cylicocyclus nassatus, Cyathostomum pateratum, Cyathostomum coronatum, Cyathostomum catinatum, Cylicostephanus longibursatus, and Cylicostephanus goldi of a BZ-resistant cyathostomin population were characterized using specific primers. The cDNA sequence of each species spans 1,429 bp, encoding a protein of 448 amino acids. The interspecific identities are 95.2-99.6% at the nucleotide and 98.7-100.0% at the peptide level. The comparison of the amino acid sequences of individuals isolated from the BZ-resistant cyathostomin population with those from individuals of Cc. nassatus, Cy. coronatum, Cy. pateratum, and Cy. catinatum of a BZ-susceptible one showed differing amino acids in 11 positions. The commonness of a phenylalanine to tyrosine mutation at position 167 in all the 6 cyathostomin species isolated from a BZ-resistant population suggests its involvement in the molecular mechanism in BZ resistance.     

Role of inducible nitrogen oxide synthase in benzene-induced oxidative DNA damage in the bone marrow of mice

We investigated the interaction of BZ and lipolysaccharide (LPS), a well-known inflammation-promoting agent, in wild-type and inducible nitrogen oxide synthase (iNOS) knockout mice. BZ generated DNA strand breaks (SB) in the liver of both wild-type and iNOS-deficient mice. In the bone marrow (BM) BZ and LPS generated SB only in wild-type mice. The effects were additive, suggesting that both a redox cycling and an iNOS-dependent pathway may be involved. Formamidopyrimidine DNA glycosylase sensitive sites were elevated by BZ in the BM in both types of mice, whereas endonuclease III sensitive sites were not affected by any treatment. Since BZ is associated with leukemia in humans, it suggests that oxidative DNA base damage rather than SB may be important in the development of leukemia.     

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.     

The heterogeneity and nucleotide modulation of cholinergic muscarinic receptors are restored by poly (ethylene) glycol-6000 precipitation of solubilized atrial receptors

The membrane bound muscarinic receptor exhibits heterogeneity of binding sites for agonists that are modulated by guanine nucleotides. These properties are generally lost upon receptor solubilization by several detergents, but may be recovered after rapid detergent removal by poly (ethylene) glycol-6000 precipitation of the digitonin plus cholate solubilized atrial muscarinic receptor. The results are discussed in terms of muscarinic receptor interactions in the intact membrane.