SEQUESTRATION OF GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS ON CLATHRIN-COATED VESICLES DURING CHRONIC BENZODIAZEPINE ADMINISTRATION IN-VIVO

Chronic administration of benzodiazepine agonists produces behavioral tolerance. For induction of tolerance, the use-dependent down-regulati on of gamma-aminobutyric acid(A) (GABA(A))/ benzodiazepine receptors i s a potential cellular mechanism. We previously identified GABA(A) rec eptors on clathrin-coated vesicles from rat brain, suggesting that: su rface receptors can be internalized via endocytosis. To examine a role for coated vesicles in GABA(A) receptor down-regulation in vivo, frac tions were obtained from mouse brain microsomes through density centri fugation and treatment with 0.1% Triton X-100. This coated vesicle pre paration was enriched in clathrin subunits and clathrin light-chain ki nase and had twice the level of [H-3]flunitrazepam binding as did vesi cles not exposed to Triton. Adult mice were treated with lorazepam (2 mg/kg/day) for 7 days via osmotic minipump, achieving a serum level of 103 +/- 8.9 ng/ml. The level of flunitrazepam bound to coated vesicle s was increased by 83 +/- 13% in the lorazepam-treated mice compared w ith vehicle-treated controls. The B-max value for [H-3]flunitrazepam b inding to synaptic membranes from lorazepam-treated animals was 33 +/- 4% lower than that of controls. The amount of GABA(A) receptor alpha- 1 subunits, as quantified by Western blotting, followed a similar patt ern. Relative to controls, immunoreactivity for alpha-1 subunits in co ated vesicles from lorazepam-treated mice was increased by 60.0 +/- 10 .3%, whereas that in synaptic membranes declined by 12 +/- 6%. These r esults indicate that lorazepam-dependent GABA(A) receptor sequestratio n occurs in mouse brain. Furthermore, it is suggested that this seques tration may play a role in GABA(A) receptor down-regulation in vivo.