Identification of GABAA/benzodiazepine receptors on clathrin-coated vesicles from rat brain.
To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABAA/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [3H]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 [3H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [3H]flunitrazepam to coated vesicles yielded a KD value of 21 +/- 4.7 nM and a Bmax value of 184 +/- 28 fmol/mg. The KD 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 Bmax 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 GABAA/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.
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
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D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002966:Clathrin / Q000378:metabolism*; D002998:Clonazepam / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D005445:Flunitrazepam / Q000378:metabolism; D008854:Microscopy, Electron; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D002966:Clathrin; D011963:Receptors, GABA-A; D002998:Clonazepam; D005445:Flunitrazepam