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

The pentameric gamma-aminobutyric acid type A receptors (GABA(A)Rs) 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 GABA(A)Rs are arranged around a central pore in the order beta-alpha-beta-alpha-gamma. In the outer third of the transmembrane domain (TMD) drugs may bind to five homologous intersubunit binding sites. Etomidate binds between the pair of beta - alpha subunit interfaces (designated as (beta(+)/alpha(-)) and R-mTFD-MPAB binds to an alpha(+)/beta(-) and an gamma(+)/beta(-) subunit interface (a beta(-) 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 pTED-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 pTFDdi-iPr-BnOH may bind between the alpha(+)/beta(-) and alpha(+)/gamma(-) subunits in the TMD, making it an alpha(+) ligand. The latter alpha(+)/gamma(-) 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 GABA A Rs. (C) 2020 Elsevier Masson SAS. All rights reserved.