Differences in the Biophysical Properties of the Benzodiazepine/γ-Aminobutyric Acid Receptor Chloride Channel Complex in the Long-Sleep and Short-Sleep Mouse Lines

Significant differences in the thermal stability of benzodiazepine receptors were found in cerebral cortical membranes prepared from the long-sleep (LS) and short-sleep (SS) selected mouse lines. Thus, benzodiazepine receptors from LS mice were heat inactivated (55 degrees C) at a significantly faster rate than those from SS mice. Although gamma-aminobutyric acid (GABA) reduced the rate of heat inactivation in both lines, the more rapid rate of inactivation in the LS line was maintained. Furthermore, the potency of GABA to enhance [3H]flunitrazepam binding decreased threefold in membranes from LS mice as the incubation temperature was increased from 0 degrees to 37 degrees C, but was unaltered in membranes from SS mice. These differences in the biophysical properties of the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex"), together with a higher KD for t-[35S]butylbicyclophosphorothionate in membranes from LS compared to SS mice, suggest that the supramolecular complex may modulate the differential sensitivity to some depressants and convulsants in these lines.     

Identification and characterization of a pregnane steroid recognition site that is functionally coupled to an expressed GABAA receptor

Conclusion Based on the pharmacological and biochemical evidence to date, especially that derived from the recombinantly expressed receptor studies, the suggestion that a novel GBRC-linked steroid recognition site exists becomes a cogent argument. The high affinity of the steroid site for certain naturally occurring metabolites of progesterone and glucocorticoids favors a physiologic role for these steroids in the regulation of brain excitability. Clearly, investigations of such a regulatory role is warranted. If present, it provides an important example of endocrine control of a major inhibitory neurotransmitter in the CNS. Moreover, as we gain a greater understanding of the molecular organization of the GBRC, the putative steroid site provides a novel target for the rational design of therapeutic agents for the treatment of anxiety, epilepsy, and insomnia.Special issue dedicated to Dr. Eugene Roberts.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Antiproliferative Action of Benzodiazepines in Cultured Brain Cells Is Not Mediated Through the Peripheral-Type Benzodiazepine Acceptor

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.     

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism, 1H NMR and molecular dynamics simulation

The conformation of a benzodiazepine-like decapeptide corresponding tothe YLGYLEQLLR fragment of a casein has been examined in a sodium dodecylsulfate micellar medium using circular dichroism, two-dimensional¹H NMR spectroscopy and restrained molecular dynamicssimulation. The decapeptide adopts an amphipathic 3₁₀-helicoidstructure in which theE⁶···R¹⁰ ionic bridgestabilizes the C-terminus.     

Potentiation of γ-Aminobutyric Acid-Mediated Chloride Flux by Pentobarbital and Diazepam but Not Ethanol

The influx of 36Cl- into cerebral cortical and cerebellar microsacs from ICR mice and Sprague-Dawley rats was studied in incubations lasting 3 s, 500 ms, or 21 ms. In the 3-s assay, 10-40 mM ethanol did not affect either basal or gamma-aminobutyric acid (GABA)-mediated Cl- flux, at any GABA concentration tested. Only at a concentration of 600 mM did ethanol potentiate Cl- flux in both mouse and rat preparations. Ethanol (20 mM) also did not affect the significant potentiation of GABA-mediated flux produced by 50 microM pentobarbital or 2 microM diazepam in ICR mouse microsacs. In 21- and 500-ms incubations (quench-flow method), 50 microM pentobarbital significantly potentiated GABA-mediated Cl- flux in rat cortical microsacs, but 10-50 mM ethanol did not. These studies suggest that some as yet unrecognized factor is essential for ethanol enhancement of GABA-mediated Cl- flux, as reported by others in brain homogenates and in tissue culture.     

New inhibitors of fungal 17β-hydroxysteroid dehydrogenase based on the [1,5]-benzodiazepine scaffold

The synthesis and activity of a new series of non-steroidal inhibitors of 17β-hydroxysteroid dehydrogenase that are based on a 1,5-benzodiazepine scaffold are presented. Their inhibitory potential was screened against 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl), a model enzyme of the short-chain dehydrogenase/reductase superfamily. Some of these compounds are potent inhibitors of 17β-HSDcl activity, with IC₅₀ values in the low micromolar range and represent promising lead compounds that should be further developed and investigated as inhibitors of human 17β-HSD isoforms, which are the enzymes associated with the development of many hormone-dependent and neuronal diseases.     

Neurosteroids, GABAA receptors, and escalated aggressive behavior

Aggressive behavior can serve important adaptive functions in social species. However, if it exceeds the species-typical pattern, it may become maladaptive. Very high or escalated levels of aggressive behavior can be induced in laboratory rodents by pharmacological (alcohol-heightened aggression), environmental (social instigation), or behavioral (frustration-induced aggression) means. These various forms of escalated aggressive behavior may be useful in further elucidating the neurochemical control over aggression and violence. One neurochemical system most consistently linked with escalated aggression is the GABAergic system, in conjunction with other amines and peptides. Although direct stimulation of GABA receptors generally suppresses aggression, a number of studies have found that positive allosteric modulators of GABAA receptors can cause increases in aggressive behavior. For example, alcohol, benzodiazepines, and many neurosteroids are all positive modulators of the GABAA receptor and all can cause increased levels of aggressive behavior. These effects are dose-dependent and higher doses of these compounds generally shift from heightening aggressive behavior to being sedative and anti-aggressive. In addition, these modulators interact with each other and can have additive effects on the GABAA receptor and on behavior, including aggression. The GABAA receptor is a heteropentameric protein that can be constituted from various subunits. It has been shown that subunit composition can affect sensitivity of the receptor to some modulators and that subunit composition differentially affects the sedative vs anxiolytic actions of benzodiazepines. Initial studies targeting alpha subunits of the GABAA receptor point to their significant role in the aggression-heightening effects of alcohol, benzodiazepines, and neurosteroids.     

Benzodiazepine-sensitive GABA(A) receptors limit the activity of the NMDA/NO/cyclic GMP pathway: a microdialysis study in the cerebellum of freely moving rats

In the cerebellum, infusion of NMDA (200 microM) for 20 min evoked a marked (200%) increase of extracellular cyclic GMP (cGMP) levels. The selective GABA(A) receptor agonist muscimol (0.01-100 microM) was able to counteract the NMDA effect with an EC(50) of 0.65 microM; the inhibitory effect of muscimol (10 microM) was prevented by bicuculline (50 microM). Diazepam (10 microM) significantly potentiated the muscimol (1 microM) inhibition; furthermore, when coinfused with 0.1 microM muscimol (a concentration not affecting, on its own, the cGMP response to NMDA), diazepam (10 microM) reduced the NMDA effect. Similar results were obtained with zolpidem (0.1-1 microM). Finally, local infusion of the benzodiazepine site antagonist flumazenil (10 microM), together with muscimol and diazepam, almost completely restored the effect of NMDA on extracellular cGMP levels. It is concluded that GABA(A) receptors potently control the NMDA/nitric oxide/cGMP pathway in the cerebellum in vivo. In terms of the alpha subunit composition, we can deduce that the cerebellar GABA(A) receptor does not contain alpha(6) or beta(4) subunits because it is diazepam-sensitive. Moreover, the observation that zolpidem is active at a rather low concentration, in combination with localization studies present in the literature, tend to exclude the presence of alpha(5) subunits in the receptor composition and suggest the involvement of an alpha(1) subunit.     

Structural Requirements for Ligand Interactions at the Benzodiazepine Recognition Site of the GABAA Receptor

Abstract: His¹⁰¹ of the GABA_A receptor α1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [³H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant α subunits were coexpressed with the rat β2 and γ2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [³H]Ro 15-4513 bound to all mutant receptors with equal or greater affinity than to the wild-type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15-1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15-1788 and Ro 15-4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect.