Loop-F of the α-subunit determines the pharmacologic profile of novel competitive inhibitors of GABAA receptors

Abstract The neurotransmitter γ-amino butyric acid (GABA) has a fundamental role in CNS function and ionotropic (GABA A ) receptors that mediate many of the actions of GABA are important therapeutic targets. This study reports the mechanism of action of novel GABA A antagonists based on a tricyclic oxazolo-2,3-benzodiazepine scaffold. These compounds are orthosteric antagonists of GABA on heteropentameric GABA A receptors of αxβ2γ2 configuration expressed in HEK293 cells. In silico modelling predicted that the test compounds docked in the GABA binding-pocket and would interact with amino-acid residues in the α- and β-subunit interface that are known to be important for the binding of GABA. Intriguingly, optimal docking also required an interaction with the non-conserved amino-terminal segment of Loop-F of the α–subunit. Testing of a compound with altered regiochemistry of the oxazolone moiety supported the model with respect to the conserved GABA-interacting residues in vitro as well as in vivo . The prediction regarding loop-F was examined by replacing the amino-terminal variable segment of loop-F of the α5-subunit with the corresponding residues in the α1- and α2-subunits. When tested with the novel inhibitors, the receptors formed by the modified α5-subunits displayed the pharmacologic phenotype of the source of loop-F. In summary, these data show that the variable amino-terminal segment of loop-F of the α-subunit determines the pharmacologic selectivity of the novel tricyclic inhibitors of GABA A receptors.