Agonist‐Dependent Internalization of γ‐Aminobutyric Acid A /Benzodiazepine Receptors in Chick Cortical Neurons

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012‐S with 4‐sulfophenylisothiocyanate. The resulting N ‐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 (IC 50 = 9.8 ± 2.9 n M ) for displacement of [ 3 H] flunitrazepam from intact chick cortical neurons was similar to that of 1012‐S (IC 50 = 4.0 ±0.3 n M ). However, at concentrations from 0.1 to 10 μ M , 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 ±‐100, a result suggesting that ∼7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1–4 h at 37°C) of neurons with 100μ M γ‐aminobutyric acid (GABA) or 100 n M clonazepam had little effect on the level of [ 3 H] flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 m M GABA increased the level of intracellular sites by 154–176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABA A receptor antagonist R 5135. The results suggest that SPTC‐1012S can be used as a probe to study the internalization of the GABA A /benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.