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Flumazenil decreases surface expression of 42 GABAA receptors by increasing the rate of receptor internalization

Kuver, A;Smith, SS;

Increases in expression of 4 GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3-OH-5[]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether 4 trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces 4 expression in vivo. We first determined that flumazenil (100 nM-100 M, IC50=1 M) acted as a negative modulator, reducing GABA (10 M)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant 42 expressed in HEK-293 cells. Surface expression of recombinant 42 receptors was detected using a 3XFLAG reporter at the C-terminus of 4 (4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 M)+THP (100 nM). Flumazenil (10 M) decreased surface expression of 4F by 60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of 42 after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of 42 were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of 42 expression using flumazenil.