Abstract

Specific binding of (+)-[³H]bicuculline methiodide ([³H]BCM) to synaptic membranes of rat cerebellum, which most likely represents an interaction with the γ-aminobutyric acid (GABA) receptor, has been characterized further. Keeping the membranes stored frozen prior to the binding assay, as routinely done, slightly reduced (10-20%) the capacity of [³H]BCM specific binding compared with freshly prepared membranes, and had a negligible effect on the affinity of BCM or GABA for the [³H]BCM binding site, as shown by their K_i values is competing for [³H]BCM specific binding: for BCM, K_i fresh = 270 ± 25 nM, and K_i frozen = 218 ± 21 nM; for GABA, K_i fresh = 490 ± 50 nM, and K_i frozen = 420 ± 55 nM. Specific [³H]BCM binding was saturable, with an apparent dissociation constant (K_d) of 380 ± 20 nM. The maximal amount of specifically bound [³H]BCM was 4.5 ± 0.2 pmoles/mg of protein. The amount of [³H]BCM specifically bound was proportional to protein concentration and showed a broad pH optimum (pH 7-9). Similar amounts of [³H]BCM were specifically bound at 25° and 37°. Equilibrium between the specific binding sites and [³H]BCM was reached within 10 min. Incubation of the membranes at temperatures above 37° or with Triton X-100 resulted in a marked decrease of [³H]BCM specific binding. Specific binding of [³H]BCM was enhanced in the presence of SCN^-, I^-, or ClO₄^- in the incubation medium, amounting to as much as 50% of total binding. The results are compatible with a GABA receptor model with two binding sites, represented by specific [³H]GABA binding and specific [³H]BCM binding, respectively. The binding sites may reflect two conformational states of the GABA receptor, an agonist and an antagonist conformation.