Abstract: | Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABAA receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [3H]muscimol, a GABAA-specific agonist, [3H]flunitrazepam, a benzodiazepine agonist, and [35S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABAA receptor channels. Under standard conditions, the binding level of [3H]muscimol, [3H]flunitrazepam, and [35S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [3H]flunitrazepam binding site with a KD value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [3H]flunitrazepam and [35S]TBPS binding assays. For [3H]flunitrazepam binding to synaptic membranes, GABA gave an EC50=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [3H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [35S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABAA receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABAA receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABAA receptor β2 and α1 subunits. Thus, in comparison to GABAA receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABAA receptor uncoupling. |