CLATHRIN-COATED VESICLES FROM BOVINE BRAIN CONTAIN UNCOUPLED GABA(A) RECEPTORS

Clathrin-coated vesicles are thought to be a vehicle for the sequestra tion of GABA(A) receptors. For coated vesicles from bovine cerebrum, w e examined the binding properties of [H-3]muscimol, a GABA(A)-specific agonist, [H-3]flunitrazepam, a benzodiazepine agonist, and [S-35]t-bu tylbiocyclophosphorthionate (TBPS), a ligand for GABA(A) receptor chan nels. Under standard conditions, the binding level of [H-3]muscimol, [ H-3]flunitrazepam, and [S-35]TBPS to coated vesicles represented 12.3 +/- 1.8%, 7.9 +/- 1%, and 10.2 +/- 1.8%, respectively, of that in crud e synaptic membranes. Coated vesicles showed a single [H-3]flunitrazep am binding site with a K-D value (12 nM) which was 9-fold that for syn aptic membranes. The allosteric coupling between binding sites was mea sured by the addition of GABA to [H-3]flunitrazepam and [S-35]TBPS bin ding assays. For [H-3]flunitrazepam binding to synaptic membranes, GAB A gave an EC50 = 2.0 mu M and at saturation (100 mu M) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonis t SR95531. In contrast, neither GABA nor SR95531 had a significant eff ect on [H-3]flunitrazepam binding to CCVs, indicating that the alloste ric interaction between GABA and benzodiazepine binding sites is aboli shed. Likewise, GABA displaced nearly all of the [S-35]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, i ndicating that coupling between the GABA binding sites and chloride ch annel is also impaired. Thus GABA(A) receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The prese nce of major GABA(A) receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic me mbranes, CCVs contained 110 +/- 14% and 29.5 +/- 3.8%, respectively, o f the immunoreactivity for GABA(A) receptor beta 2 and alpha 1 subunit s. Thus, in comparison to GABA(A) receptors on synaptic membranes, tho se on CCVs have a reduced alpha 1/beta 2-subunit ratio. It may be sugg ested that a selective decline in the content of alpha 1 subunits in c oated vesicles could in part account for GABA(A) receptor uncoupling. (C) 1997 Elsevier Science B.V.