High Specific Binding of [³H]GABA and [³H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Abstract

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.

m‐Sulfonate Benzene Diazonium Chloride: A Powerful Affinity Label for the γ‐Aminobutyric Acid Binding Site from Rat Brain

“…We found that [3H]muscimol was a radioligand of choice for binding studies on the GABA receptor of filter-adsorbed membranes, because it displayed a negligible adsorption to GF/B filters and it associated rapidly to the receptor sites at 25°C. Our experimental conditions allowed specific [3H]muscimol binding to fulfill the following criteria: (a) favorable specific versus nonspecific binding ratio; (b) linear relationship between specific binding and the amount of adsorbed membranes; (c) saturability; and (d) drug affinity properties in agreement with those determined for specific [3H]muscimol binding in solution (Iversen et al, 1978;Horng and Wong, 1979;Olsen, 1981;Quinn and Cagan, 1982;Fisher et a]., 1986). This [3H]muscimol filter binding assay is rapid, reliable, and easy to handle.…”

Taurine allosterically modulates flunitrazepam binding to synaptic membranes

“…All values are corrected for nonspecific binding measured in the presence of l p M unlabeled clonazepam. Ligand bound to membrane was separated from free ligand by rapid filtration through Whatman GFiB filters as previously described (Quinn and Cagan, 1982). Filters were rinsed with 10 ml buffer and radioactivity retained on the filter was measured by liquid scintillation spectrometry.…”

Decreased High‐Affinity Binding of [3H]Muscimol to Cerebral Synaptic Membranes of Scrapie‐Infected Mice

“…CSM protein was measured by the Lowry method (Lowry et al, 1951), with bovine serum albumin as standard. Binding was measured by a modification of a previously described rapid filtration method (Quinn and Cagan, 1982). The standard incubation mixture contained 2 nM [3H]muscimol (10 Ci/mmol) and 200-300 pg CSM protein in a final volume of 1.1 ml of 50 mM Tris-citrate, pH 7.1.…”