Kinetics of [3H]muscimol binding to the GABAA receptor in bovine brain membranes

Abstract

The binding of the GABA receptor agonist [3H]muscimol to membrane preparations from bovine cerebral cortex has been investigated in equilibrium and kinetic experiments. Equilibrium binding curves are biphasic and suggest that [3H]muscimol binds to both high-affinity (Kd approximately 10 nM) and low-affinity (Kd approximately 0.5 microM) sites. Binding to each class of sites is inhibited by GABA and by the specific GABAA receptor antagonist bicuculline. The kinetics of [3H]muscimol binding have been measured by using both manual filtration assays and an automated rapid filtration technique which permits the measurement of ligand dissociation on subsecond time scales. Association and dissociation curves are biphasic at all concentrations of [3H]muscimol studied, even under conditions of low receptor saturation when no significant occupancy of the low-affinity sites would be expected. These results cannot be simply explained by the presence of two populations of binding sites in the membrane preparations but suggest the existence of two forms of the monoliganded receptor. Dissociation constants for these two forms have been estimated to be 16 and 82 nM at 23 degrees C. At higher ligand concentrations, kinetic measurements have suggested that the binding of [3H]muscimol to low-affinity sites is accompanied by a slow conformational change of the receptor-ligand complex.

Tyrosine 62 of the γ-Aminobutyric Acid Type A Receptor β2 Subunit Is an Important Determinant of High Affinity Agonist Binding

“…Equilibrium (33) and carried out according to the method as described previously (4). Use of this system, in which the filter washing step was reduced to 0.…”

“…The GABA A R carries binding sites for a number of therapeutic agents including the benzodiazepines, barbiturates, neurosteroids, some general anesthetics, and possibly also alcohol (1). In brain membranes, there are at least two classes of binding sites for the endogenous neurotransmitter, which differ by more than an order of magnitude in their affinity for GABA or its structural analogues (2)(3)(4). This heterogeneity in binding was originally thought to reflect the diversity of GABA A R subtypes in brain tissue.…”

Agonist Binding to the Torpedo Acetylcholine Receptor. 1. Complexities Revealed by Dissociation Kinetics

“…Aliquots (0.35 mL) of nAcChR-enriched membranes were dialyzed against 0.35 mL of radiolabeled ligand solution with rocking for 6 h at 4°C. Total and free ligand concentrations were measured by taking duplicate 50 µL samples of each, adding 4 mL of hydrofluor (National Diagnostics) (DuPont, 1984;Agey & Dunn, 1989). DNPP-treated membranes were equilibrated with [ 3 H]AcCh or [ 3 H]SdCh by incubation for 30 min at room temperature.…”

Functional Consequences of the Loss of High Affinity Agonist Binding to γ-Aminobutyric Acid Type A Receptors

“…The number of agonist-binding sites on a single GABA A R has not been unequivocally defined, owing in large part to the inherent complexity of this receptor family. Early binding studies using native brain membranes suggested that there were at least two classes of binding sites of high (K D ϭ 10 -30 nM) and low (K D ϭ 0.1-1.0 M) affinity (11,13). Although these observations may have arisen from the heterogeneity of GABA A Rs that are now known to exist in mammalian brain, more recent studies using recombinant, presumably homogeneous, receptor subtypes suggest that individual receptors carry both high and low affinity sites for agonists (10,20).…”

“…Radioligand binding studies have revealed the existence of (at least) two classes of agonist recognition sites on a single GABA A R (10), which differ in affinity by more than 1 order of magnitude (see also Refs. [11][12][13]. By using the recombinant rat ␣1␤2␥2 receptor expressed in tsA201 cells, we found two classes of binding sites for [ 3 H]muscimol with K d values of 8.1 and 430 nM (10).…”