Abstract

The binding of radioactive γ-aminobutyric acid (GABA) to receptor-like sites in mammalian brain membranes was analyzed by computer for comparison with models which might explain the observed apparent heterogeneity of ligand binding. The best fit was obtained with two independent binding sites. Binding was measured by centrifugation, using thoroughly washed, frozen, and thawed membranes without detergent treatment. Assays were carried out at 0° under sodium ion-free conditions which have previously been shown to allow detection only of those binding sites having the chemical specificity and other properties expected of receptor sites for the neurotransmitter GABA. Quantitative analysis of binding curves for several brain regions, subcellular fractions, and species revealed the general presence of two affinity classes for GABA receptors, one with K_D Of 13 ± 6 nM (B_max = 0.33 pmole/mg of protein in bovine cortex) and the other with K_D of 300 ± 150 nM (B_max 1.8 pmole/mg of protein in bovine cortex). The two-site model fit the data better than did models with one site, three sites, or negative cooperativity, but the fit to the mobile receptor-effector coupling hypothesis was almost as good as that of the two-site model. Consistent with the heterogeneity in equilibrium binding data, heterogeneity was also observed for association and dissociation rates of ligand binding and for rates of thermal inactivation of binding activity. The rate of heat denaturation was biphasic, with earlier times corresponding to selective loss of one of the two binding affinity subpopulations. Kinetics studies revealed two subpopulations of binding sites with respect to on- and off-rates. A slow component with k₊₁ = 1.9 x 10⁷ M^-1 min^-1, and k_-1 = 0.2 min^-1, K_D = 11 nM, and low B_max corresponded to the high-affinity component of the equilibrium binding curve, and a rapidly dissociating population with lower affinity and higher B_max corresponded to the lower-affinity component of the equilibrium binding curve. Independent IC₅₀ values for the high-affinity and low-GABA site were determined for a series of analogues. All analogues tested were more effective inhibitors of the high-affinity GABA sites, and a marked similarity in the relative drug potency for the two sites was observed. Thus the heterogeneity of GABA binding would appear to involve two discrete populations of receptors on the grounds of noninterconvertible heterogeneity under the conditions tested, whereas the similarity in the drug specificity of the two populations would be more consistent with a model involving multiple coupled or conformational states of a single receptor.