Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABAA, Receptors

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABAA receptors via their extracellular alpha x+/gamma 2- (x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous alpha+/beta- interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure activity relationship models for benzodiazepine site activity. Considering all possible a+/beta- and alpha+/gamma- interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity alpha 1+/gamma 2- site with the homologous alpha 1+/beta 3- site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the gamma 2- subunit into the beta 3- subunit, resulting in a quadruple mutant beta 3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant alpha 1 beta 3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous alpha+/beta- sites.