title: Agonist-dependent internalization of gamma-aminobutyric acidA/benzodiazepine receptors in chick cortical neurons.
abstract: An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012-S with 4-sulfophenylisothiocyanate. The resulting N-(4-sulfophenyl)-thiocarbamoyl derivative of 1012-S (SPTC-1012S) was purified by reverse-phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC-1012S (IC50 = 9.8 +/- 2.9 nM) for displacement of [3H]flunitrazepam from intact chick cortical neurons was similar to that of 1012-S (IC50 = 4.0 +/- 0.3 nM). However, at concentrations from 0.1 to 10 microM, 1012-S was consistently more efficacious than SPTC-1012S, a finding indicating that 6-8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton X-100, a result suggesting that approximately 7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1-4 h at 37 degrees C) of neurons with 100 microM gamma-aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H]flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154-176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC-1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.
authors.affiliation: Department of Biochemistry and Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030.
authors.forename: M H
authors.initials: MH
authors.lastname: Tehrani
authors.forename: E M
authors.initials: EM
authors.lastname: Barnes
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D002642:Chick Embryo; D002998:Clonazepam / Q000494:pharmacology; D005445:Flunitrazepam / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009994:Osmolar Concentration; D011963:Receptors, GABA-A / Q000378:metabolism / Q000502:physiology*; D012044:Regression Analysis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D002998:Clonazepam; D005445:Flunitrazepam; C060004:1012S