Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the crosslinker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.

Benzodiazepines probably exert their anxiolytic, hypnotic, and anticonvulsant effects by interacting with brain-specific high-affinity benzodiazepine receptors. In searching for possible endogenous ligands for these receptors, we have purified a compound 107-fold from human urine by extractions, treatment with hot ethanol, and column chromatography. The compound was identified as ,-carboline-3-carboxylic acid ethyl ester (IIc) by mass spectrometry, NMR spectrometry, and synthesis; HIc was also isolated from brain tissues (20 ng/g) by similar procedures. Very small concentrations of TIc displaced [3H]diazepam completely from specific cerebral receptors, but not from liver andkidney binding sites; the concentration causing 50% inhibition of specific [3H]diazepam binding (IC50) was 4-7 nM compared to ca. 5 nM for the potent benzodiazepine lorazepam. Specific binding sites for quinuclidinyl benzilate, naloxone, spiroperidol, serotonin, muscimol, and WB 4101 were not affected by IIc. In contrast to benzodiazepines, I1c exhibits "mixed type" competitive inhibition of forebrain benzodiazepine receptors (negative cooperativity). We surmise that an endogenous ligand for benzodiazepine receptors may be a derivative of P-carboline-3-carboxylic acid.[3H]Diazepam and [3H]flunitrazepam bind specifically and with high affinity to brain membranes from all higher vertebrates, including man (1-5). Binding is brain specific (6), correlated to pharmacological potency (1, 7-10), located on neurons (11-17), distributed unevenly in the brain (6), and slightly sensitive to seizures and stressful conditions (18,19 5.5. Further impurities were removed by washing twice with 0.05 vol of borate buffer (100 mM, pH 9) and three times with 0.05 vol of aqueous NH3 (100 mM). All aqueous phases were washed twice in counter current fashion with ethyl acetate to decrease losses. The combined ethyl acetate phase was evaporated to dryness and the residue was dissolved in 300 ml of 0.18-0.25 M HCI and washed with 500 ml of diethyl ether, which removed large amounts of impurities. The active fraction was then extracted twice with 500 ml of diethyl ether at pH 5-5.5. After evaporation the brown-orange waxy residue was dissolved in 3 ml of 50% ethanol and chromatographed on a Sephadex LH-20 column [1.5 X 84 cm, elution volume (Ve) = 144 ml] in 50% ethanol. The active fraction (y-fraction) was eluted in a volume of [15][16][17][18][19][20][21][22][23][24][25]

The gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex, which is composed of distinct proteins embedded in the neuronal plasma membrane, is important for several effects of benzodiazepines, including protection afforded against convulsions. During structural modification of ethyl beta-carboline-3-carboxylate an agent was discovered which has high affinity for brain benzodiazepine receptors but which is a potent convulsant. Also in contrast to benzodiazepines, this type of benzodiazepine receptor ligand favors benzodiazepine receptors in the non-GABA-stimulated conformation, which may explain the convulsive properties.

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The affinities for the benzodiazepine binding site of the GABA(A) receptor of 21 flavonoids have been studied using [(3)H]flumazenil binding to rat cortical membranes in vitro. We show that flavonoids with high affinity for the benzodiazepine receptor in vitro spanning the whole efficacy range from agonists (1q) to inverse agonists (1l) can be synthesized. The receptor binding properties of the flavonoids studied can successfully be rationalized in terms of a comprehensive pharmacophore model recently developed by Cook and co-workers (Drug Des. Dev. 1995, 12, 193-248), supporting the validity of this model. However, in contrast to the requirement by the model that an interaction with the hydrogen bond-accepting site A2 is necessary for compounds to display inverse agonistic activity, 6-methyl-3'-nitroflavone (1l), which cannot engage in such an interaction, nevertheless displays inverse agonism. The analysis of the binding affinities of 3'- and 4'-substituted flavones in terms of the pharmacophore model has yielded new information for the further development of the pharmacophore model.