A potent photoreactive general anesthetic with novel binding site selectivity for GABAA receptors

The pentameric γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory ligand-gated ion channels in the central nervous system. They mediate diverse physiological functions, mutations in them are associated with mental disorders and they are the target of many drugs such as general anesthetics, anxiolytics and anti–convulsants. The five subunits of synaptic GABAARs are arranged around a central pore in the order β-α-β-α-γ. In the outer third of the transmembrane domain (TMD) drugs may bind to five homologous intersubunit binding sites. Etomidate binds between the pair of β – α subunit interfaces (designated as β+/α–) and R–mTFD-MPAB binds to an α+/β– and an γ+/β– subunit interface (a β– selective ligand). Ligands that bind selectively to other homologous sites have not been characterized. We have synthesized a novel photolabel, (2,6-diisopropyl-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanol or pTFD-di-iPr-BnOH). It is a potent general anesthetic that positively modulates agonist and benzodiazepine binding. It enhances GABA–induced currents, shifting the GABA concentration-response curve to lower concentrations. Photolabeling–protection studies show that it has negligible affinity for the etomidate sites and high affinity for only one of the two R–mTFD-MPAB sites. Exploratory site–directed mutagenesis studies confirm the latter conclusions and hint that pTFD-di-iPr-BnOH may bind between the α+/β– and α+/γ– subunits in the TMD, making it an α+ ligand. The latter α+/γ– site has not previously been implicated in ligand binding. Thus, pTFD-di-iPr-BnOH is a promising new photolabel that may open up a new pharmacology for synaptic GABAARs. [Display omitted] •PTFD-di-iPr-BnOH is a potent general anesthetic and enhances GABA’s actions.•It photo-incorporates into intersubunit sites in the GABA(A)R’s transmembrane domain.•Photo-protection shows it binds at either the γ+/β– or the α+/β– subunit interface.•Similarly, it does not bind in the β+/α– subunit interfaces (etomidate sites).•Site–directed mutagenesis suggests the α+/γ– interface is another site.