Clathrin-coated vesicles from bovine brain contain uncoupled GABA A receptors

Abstract

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [ 3H]muscimol, a GABA A-specific agonist, [ 3H]flunitrazepam, a benzodiazepine agonist, and [ 35S] t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA A 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 K D 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 EC 50=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 GABA A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA A 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 GABA A receptor β2 and α1 subunits. Thus, in comparison to GABA A 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 GABA A receptor uncoupling.