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 Ki values is competing for [3H]BCM specific binding: for BCM, Ki fresh = 270 ± 25 nM, and Ki frozen = 218 ± 21 nM; for GABA, Ki fresh = 490 ± 50 nM, and Ki frozen = 420 ± 55 nM. Specific [3H]BCM binding was saturable, with an apparent dissociation constant (Kd) 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.
ACKNOWLEDGMENT We thank Dr. W. Haefely for critical reading of the manuscript.
- Copyright © 1978 by Academic Press, Inc.
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|