Identification of GABA A /Benzodiazepine Receptors on Clathrin‐Coated Vesicles from Rat Brain

To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABA A /benzodiazepine receptors, we have studied the distribution and properties of clonazepam‐displaceable binding of [ 3 H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin‐coated vesicles. Homogeneity of the coated‐vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light‐chain kinase. Vesicles exhibiting specific binding of [ 3 H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [ 3 H]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 synaptic membranes. This low‐affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The B max value for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. 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 removal of GABA A /benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down‐regulation that is produced by exposure to GABA and benzodiazepine agonists.