Identification of GABAA/benzodiazepine receptors on clathrin-coated vesicles from rat brain

J Neurochem. 1993 May;60(5):1755-61. doi: 10.1111/j.1471-4159.1993.tb13400.x.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding, Competitive
  • Brain / metabolism*
  • Brain / ultrastructure
  • Clathrin / metabolism*
  • Clonazepam / metabolism
  • Coated Pits, Cell-Membrane / metabolism*
  • Coated Pits, Cell-Membrane / ultrastructure
  • Female
  • Flunitrazepam / metabolism
  • Microscopy, Electron
  • Rats
  • Receptors, GABA-A / metabolism*
  • Subcellular Fractions / metabolism


  • Clathrin
  • Receptors, GABA-A
  • Clonazepam
  • Flunitrazepam