IDENTIFICATION OF GABA(A) BENZODIAZEPINE RECEPTORS ON CLATHRIN-COATEDVESICLES FROM RAT-BRAIN

To investigate the subcellular compartments that are involved in the e ndocytosis and intracellular trafficking of GABA(A)/benzodiazepine rec eptors, we have studied the distribution and properties of clonazepam- displaceable binding of [H-3]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifuga tion and gel filtration to isolate clathrin-coated vesicles. Homogenei ty of the coated-vesicle fraction was demonstrated by using electron m icroscopy and by analysis of clathrin subunits and clathrin light-chai n kinase. Vesicles exhibiting specific binding of [H-3]flunitrazepam e luted from the sieving gel as a separate peak, which was coincident wi th that for coated vesicles. Scatchard analysis of equilibrium binding of [H-3]flunitrazepam to coated vesicles yielded a K(D) value of 21 /- 4.7 nM and a B(max) value of 184 +/-28 fmol/mg. The K(D) value for coated vesicles was 12-19-fold that found with microsomal or crude syn aptic membranes. This low-affinity benzodiazepine receptor was not ide ntified on any other subcellular fraction and thus appears to be a nov el characteristic of coated vesicles. The B(max) value for coated vesi cles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, re spectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the re moval of GABA(A)/benzodiazepine receptors from the neuronal surfaces o f the rat brain. This mechanism could play a role in receptor sequestr ation and down-regulation that is produced by exposure to GABA and ben zodiazepine agonists.