Characterization of Na⁺‐Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum

Abstract

The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and post-synaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken-up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.

Substructure in the postsynaptic density of purkinje cell dendritic spines revealed by rapid freezing and etching

“…Biochemical techniques have detected a variety of proteins associated with postsynaptic densities. Several of these have an obvious relationship to synaptic function and probably are associated with the postsynaptic membrane, including receptors for glutamate (Fagg and Matus, 1984), gamma-aminobutyric acid (Wong and Horng, 1977;Lester et al, 1981;Matus and Pehling, 1981), and flunitrazepam (Carlin and Siekevitz, 1984). In addition, Kennedy and coworkers have shown that a surprisingly large portion of the protein associated with postsynaptic densities is a protein kinase (Kennedy et al, 1983;Miller and Kennedy, 1985).…”

γ‐Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site

“…Thomas and Tallman (198 1) attribute heterogeneity to lack of removal of unbound label from the membranes following labelling, and report labelling of a single protein, M, 48K, from rat brain. More recently canine cortical and cerebellar synaptosomal membranes have been reported to possess a single protein of M, 5 1 K that can be photoaffinity-labelled (Carlin and Siekevitz, 1984). Barnard et al (1984) reported that in purified bovine GABA/benzodiazepine receptor complex labelling is associated with a major protein of M, 53K.…”

The Postsynaptic Density: Constituent and Associated Proteins Characterized by Electrophoresis, Immunoblotting, and Peptide Sequencing

“…Actin, tubulin, microtubule associated proteins (MAPS), especially MAP11 Caceres et al, 1983), and calmodulin and spectrin have also been identified in the PSD fraction (Lin et al, 1980;Zagon et al, 1986). Neuroreceptor binding studies indicate major enrichment for y-aminobutyric acid (GABA) and glutamate receptors (Carlin and Siekevitz, 1984;Fagg and Matus, 1984;Wu et al, 1986~;Foster and Fagg, 1987), and Ca2+ and K+ channels (Wu et al, 1985(Wu et al, , 1986b. Specifically, the L-type Ca2+ channel and fl-adrenergic receptor have been localized in the PSD by immunocytochemistry (Strader et al, 1983;Westenbroek et al, 1990).…”