Cerebral cortical GABA and benzodiazepine binding sites in genetically seizure prone rats

Adult male and female genetically seizure-prone rats were assessed for sound-induced seizures. Heterozygous control groups were compared with mild seizure (designated GEPR 3) and severe seizure animals (GEPR 9). Groups of animals were killed and crude synaptosome fractions (P2) prepared from freshly dissected cerebral cortices. Binding sites for gamma-aminobutyric acid (GABA) were assessed by [3H]-muscimol in the absence or presence of excess GABA and/or pentobarbital. Binding sites for benzodiazepines were assessed by [3H]-flunitrazepam in the presence or absence of clonazepam. Compared to controls, GEPR 3 animals had a modest increase and GEPR 9 animals a larger increase in Bmax for both high and low affinity GABA sites, with no change in Kd. Chloride-dependent, barbiturate-enhanced GABA binding (increased Bmax) was observed in all conditions and groups. Likewise benzodiazepine binding (Bmax) increased slightly in GEPR 9 animals. There were no observed changes in binding sites for a survey of biogenic amines. Seizure-prone animals appear to have compensatory denervation-like supersensitivity for their most prominent inhibitory receptor, which may or may not be linked to the seizure event.     

Solubilization of benzodiazepine binding site from rat cortex.

The high-affinity binding site for [³H] diazepam has been solubilized from rat brain using 0.5% Lubrol-PX. Using a polyethylene glycol (PEG)-γ-globulin assay, it has been possible to demonstrate solubilization of about 60% of the binding sites in a single step. The solubilized binding site possesses a K_D of 11 nM for [³H] diazepam compared to approximately 4 nM for the membrane-bound form, and binding is to a single class of sites. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Binding of [³H] diazepam is temperature dependent and higher at 4° than 37°C. Both urea and guanidine-HC1 were capable of totally inhibiting binding, and this inhibition was partly reversible; neither sulfhydryl groups nor carbohydrate moieties seem to be important for binding. γ-Aminobutyric acid which enhanced [³H] diazepam binding to membrane fractions was without effect on the solubilized binding site.     

Endogenous effector of the benzodiazepine binding site: purification and characterization

A protein has been isolated from the small intestine and bile duct which inhibits the binding of [3H]diazepam to specific benzodiazepine binding sites on synaptosomal membranes. When ion-exchange chromatography and gel filtration chromatography are used, this protein has been purified to apparent homogeneity. "Nepenthin" has been chosen as a name for this protein, which has an approximate molecular weight of 16 000, as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. Purified nepenthin is a competitive inhibitor of [3H]diazepam binding with a Ki = 4.6 X 10(-8) M. It does not inhibit the binding of specific ligands to the enkephalin, beta-adrenergic, gamma-aminobutyrate, or dopamine binding sites in the CNS. Neither gamma-aminobutyric acid nor glycine alters the inhibition of [3H]diazepam binding by this protein. Nepenthin can be extensively treated with proteases (trypsin, chymotrypsin, and Pronase), and inhibition of diazepam binding remains stable, indicating that a lower molecular weight fragment retains activity. Antibodies raised against this purified effector have been used in in situ double antibody labeling studies with rat brain slices. These studies indicate that cells containing an immunologically similar material are present in the deep cortical region of the forebrain.     

Differential modulation by muscimol of [3H] flunitrazepam binding to benzodiazepine binding site subtypes

The effects of the GABA agonist, muscimol on [³H]flunitrazepam binding were examined in cerebellum and hippocampus regions proposed to contain different populations of benzodiazepine binding site subtypes. Quantitative analysis was made of the contribution of different components of [³H]flunitrazepam binding by utilising the selective affinities of propyl β-carboline-3-carboxylate for these sites. The influence of muscimol on each of these components was determined and the results provide clear evidence that GABA receptors interact with only some subtypes of benzodiazepine binding sites; for example, whilst the cerebellar site and the low affinity hippocampal site are influenced, the high affinity site in hippocampus appears to be quite unaffected.     

Decreased 3H-diazepam binding is a specific response to chronic benzodiazepine treatment.

Specific ³H-diazepam binding was measured in rat cortex after 7–10 days of twice daily treatment with large doses of either flurazepam or barbital. Subjective ratings of drug response each day showed that barbital treated rats experienced a degree of ataxia equal to, or greater than, that experienced by the benzodiazepine treated animals. Before performing the binding assay, the membrane preparation was washed 3 times in hypotonic buffer to remove aby residual drug. Flurazepam treatment caused a 16% decrease in maximal ³H-diazepam binding, but no change in binding affinity, confirming previous results. In contrast, barbital treatment caused no change in either binding affinity or in maximal binding. Thus, decreased diazepam binding appears to be a specific response to prolonged receptor occupation, and not part of a more generalized adaptation to chronic CNS depression.     

Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABA(A) receptors

A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed on GABA(A) receptor subtypes (alpha(1)beta(3)gamma(2s), alpha(2)beta(3)gamma(2s), alpha(4)beta(3)gamma(2s) and alpha(5)beta(3)gamma(2s)), displaying nanomolar affinities as well as selectivity for alpha1- versus alpha2- and alpha3-containing receptors by a factor of between 14 and 26.     

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.     

The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site

A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.     

NMR and density study of Co site binding by polyelectrolytes

The changes of density and chemical shifts of the water proton upon addition of CoCl₂ to aqueous solutions of tetramethylammonium salts of seven polyelectrolytes (polyphosphate, maleic acid-methylvinylether alternated copolymer, polyacrylic acid, carboxymethylcellulose of substitution degree 0.98, 1.3, 2.1, 2.65) have been measured. Assuming a negligible contribution of pseudo-contact interaction to the water proton chemical shift and a constant hyperfine constant upon displacement of water molecules by other ligands, has permitted the calculation of (i) the number of water molecules released by Co²⁺ ions upon binding and the approximate fraction of Co²⁺ ions bound with loss of water, and (ii) the total volume change upon binding and the individual contributions of counterions and polyelectrolyte charged sites to this volume change. Our results are generally in agreement with those obtained using other methods.     

Tetranitromethane modification of human high density lipoprotein (HDL3): inactivation of high density lipoprotein binding is not related to cross-linking of phospholipids to apoproteins

Treatment of human high density lipoprotein (HDL) with tetranitromethane (TNM) inhibits its binding to HDL-specific binding sites of cells and isolated membranes. The mechanism of this inhibition, however, is not known; during treatment of HDL with TNM, in addition to the expected nitration of tyrosine residues, cross-linking of lipids to apoproteins and of apoproteins to one another occurs. In order to determine whether the cross-linking of lipids to apoproteins occurs through the carbon-carbon double bonds in the acyl chains, and to determine whether the cross-linking of phospholipids to apoproteins is a possible mechanism of inhibition of binding, we have prepared a reconstituted HDL3 in which the native phospholipids were replaced with dimyristoyl phosphatidylcholine (DMPC). As a control, a reconstituted HDL3 (C-r-HDL3) was also prepared using the total apoproteins and the total lipid constituents of native HDL3. The reconstituted DMPC-containing HDL3 (DMPC-r-HDL3) was similar to native HDL3 and to C-r-HDL3 in its agarose gel electrophoretic mobility, in its chemical composition, and in its binding to rat liver plasma membranes. When treated with TNM, DMPC-r-HDL3, like the native HDL3 and C-r-HDL3, lost its ability to bind to the HDL binding sites of rat liver plasma membranes, as determined by competitive binding assays with 125I-labeled human HDL3 as the tracer. Nitrated DMPC-r-HDL3 contained only traces of phospholipids covalently linked to apoproteins, whereas 21-26% of the total phospholipids were cross-linked to apoproteins of nitrated C-r-HDL3 and nitrated native HDL3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparative and analytical separation of the zopiclone enantiomers and determination of their affinity to the benzodiazepine receptor binding site

We report the preparative separation of rac-zopiclone using malic acid as the resolving agent. Furthermore, two different methods for the analytical determination of zopiclone enantiomers by HPLC on chiral stationary phases are described. The benzodiazepine receptor binding of the isolated enantiomers was investigated. Half-maximal inhibitory concentrations of (+)- and (?)-zopiclone were 21 or 1,130 nmol/liter, respectively, indicating a more than 50 times higher affinity of the (+)-enantiomer toward the receptor. © 1993 Wiley-Liss, Inc.     

Cyclosporine increases endothelin-1 binding site density in cardiac cell membranes

When used in high concentrations, the immunosuppressive agent cyclosporine A has toxic side effects, including cardiac fibrosis and calcification. Cyclosporine-treated mice were used to investigate whether a cyclosporine dose regime which produces cardiac fibrosis and calcification alters the density of the specific binding sites for the endothelial-derived vasoconstrictor polypeptide, endothelin-1. After twenty-one days of treatment the endothelin-1 binding site density in cardiac cell membranes was increased, without any change in selectivity. This increase in endothelin-1 binding site density could contribute to the cytotoxic effects of cyclosporine.     

Endosomal density shift is related to a decrease in fusion capacity.

Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.     

Hepatic prolactin binding is rapidly altered by endotoxin in lactating mice

Endotoxin or lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, produces profound physiologic changes in most mammals. The effects of LPS on ovine prolactin (oPRL) binding by hepatic membranes of lactating mice is explored in this report. Specific 125I-oPRL binding by liver membranes from LPS-responder C3HfB/HeN mice increased two-fold within fifteen minutes of the injection of LPS, while no change was observed in the non-responder C3H/HeJ mice. Specific 125I-insulin binding did not change. Scatchard analysis of equilibrium binding of oPRL to C3HfB/HeN liver membranes indicated that within fifteen minutes of LPS injection, a receptor of differing binding affinity appears and then disappears by one hour post-injection. We propose that these rapid alterations in the specific binding of oPRL by liver membranes from LPS-injected, lactating C3HfB/HeN mice are due to the transient creation or unmasking of a novel class of PRL receptor.     

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.     

Identification of inosine as an endogenous modulator for the benzodiazepine binding site of the GABAA receptors

Previously we have reported the presence of endogenous ligands that are involved in the regulation of the binding of muscimol to the GABA binding site of the GABA_A receptors. Here, we report the presence of multiple forms of endogenous ligands in the brain which modulate the binding of flunitrazepam (FNZP) to the benzodiazepine (BZ) binding site of the GABA_A receptor. Furthermore, one of the endogenous ligands for the BZ receptors, referred to as EBZ, has been identified as inosine based on the following observations: (1) standard inosine and the EBZ have identical NMR and UV spectra; (2) the elution profile of inosine and the EBZ from a HPLC column are indistinguishable, and (3) inosine and the EBZ show identical activity in inhibiting [³H]FNZP binding.     

Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site.

As model compounds for Ni(II)-binding heparin-like compounds isolated from human kidneys (Templeton, D.M. & Sarkar, B. (1985) Biochem. J. 230 35-42.), we investigated two disaccharides--4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-2,5-anhydro- D-mannitol, disodium salt (1a), and 4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-6-O- sulfo-2,5-anhydro-D-mannitol, trisodium salt (1b)--that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate (1a) was active whereas the disulfate (1b) was inactive in a high-performance liquid chromatography (HPLC) binding assay with the tracer ions 63Ni(II) 54Mn(II), 65Zn(II), and 109Cd(II). This result is in accord with the isolation of two 67Cu(II) and 63Ni(II) binding fractions from a complete pool of nitrous-acid-derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound (1a) and the other as a tetrasulfated trisaccharide by high resolution FAB-MS, NMR and HPLC-PAD. Similarly, two synthetic disaccharides-methyl, 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-alpha-D-glucosamine, trisodium salt [IdopA2S(alpha 1,4)GlcNS alpha Me, 2a], and 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-6-O-sulfo- alpha-D-glucosamine, tetrasodium salt [IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b]--were shown to bind tracer amounts of 63Ni and 67Cu using chromatographic assays. Subsequently, 1H NMR titrations of 1a, 1b, 2a, and 2b with Zn (OAc)2 were analyzed to yield 1:1 Zn(II)-binding constants of 472 +/- 59, 698 +/- 120, 8,758 +/- 2,237 and 20,100 +/- 5,598 M-1, respectively. The values for 2a and 2b suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 1H and 13C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions.     

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.