engleski

Allosteric interactions of GABA A receptor binding sites following chronic flumazenil treatment

GABA A (gamma-aminobutyric acid A) receptors are ligand-gated ion channels composed of various polypeptide subunits. The functional and pharmacological properties of these receptors, including the potency and efficacy of the neurotransmitter GABA itself, depend on their subunit composition. GABA A receptors possess binding sites for a variety of drugs (benzodiazepines, barbiturates, neurosteroids etc). Chronic treatment with these drugs leads to regulatory changes often resulting in functional uncoupling of GABA A receptor binding sites. The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to flumazenil, the antagonist of benzodiazepine binding sites. Such studies might be important since flumazenil is not only being used acutely to reverse effects of over sedation with benzodiazepines, but is also considered for treatment of hepatic encephalopathy patients. Human embrionic kidney (HEK) 293 cells stably expressing alpha1 beta2 gamma2S subtype of GABA A receptor, the most common type of GABA A receptors found in the brain, were exposed to flumazenil (1 or 5 microM) in the presence of 1 microM GABA for 48 h. Membrane preparations (~ 100 microg protein) obtained from control and drug treated cells were incubated with increasing concentrations of [3H]flunitrazepam and [3H]TBOB to determine the maximum number (Bmax) and dissociation constant (Kd) of binding sites for benzodiazepines and convulsants. In stimulation studies the ability of GABA (1nM-1mM) to potentiate [3H] flunitrazepam (1nM) binding, and in inhibition studies the ability of GABA (1 nM - 1 mM) and diazepam (1 nM - 1 mM) in the presence of GABA (1 microM) to modulate the binding of convulsant [3H]TBOB (8 nM) was used to assess the allosteric interactions of GABA A receptor binding sites. Results: Prolonged (48 h) exposure of HEK 293 cells expressing recombinant alpha1 beta2 gamma2S GABA A receptors to flumazenil (1 and 5 microM) enhanced the Bmax of [3H] flunitrazepam and [3H]TBOB binding sites without affecting their Kd value. Addition of GABA enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil pretreated cells in a concentration-dependent manner, but there was no significant difference in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. GABA inhibited the binding of [3H]TBOB in a concentration-dependent manner, while diazepam (1nM-1mM) in the presence of 1microM GABA inhibited the binding of [3H]TBOB according to a two-site model. Approximately 40% of binding sites had an IC50 in the nanomolar, while the remaining had an IC50 in the micromolar range. No significant differences between the groups were observed in either the potency (IC50) or efficacy (Imax) of GABA and diazepam to modulate [3H]TBOB binding. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1 beta2 gamma2S GABA A receptors to flumazenil in the presence of GABA, up-regulates binding sites for benzodiazepines and convulsants, but it does not appear to affect the functional interactions of GABA A receptor binding sites, as evidenced by an unchanged ability of GABA to potentiate binding of benzodiazepines as well as by an unchanged ability of GABA and diazepam to modulate the binding of convulsants.

GABA A receptor; HEK 293 cells; chronic flumazenil treatment; [3H]flunitrazepam binding; [3H]TBOB binding

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