Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α1β3γ2 GABAAR Transmembrane Domain

GAB(A)A receptors (GAB(A)ARs) are targets for important classes of clinical agents (e. g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([H-3] azietomidate) and mephobarbital [[H-3]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid ([H-3]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the alpha 1 beta 3 gamma 2 GABA(A)R transmembrane domain at beta(+) -alpha(-) (beta(+) sites) and alpha(+) -alpha(-)/gamma(+) -beta(-) (beta(-) sites) subunit interfaces. We now use competition photolabeling with [H-3]azietomidate and [H-3] R-mTFDMPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to beta(+), while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl) methyl)-propofol] bind with similar to 10-fold higher affinity to beta(-) sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the alpha(-) -beta(-) and gamma(+) -beta(-) sites. However, we discovered four compounds that bind with different affinities to the two beta(-) interface sites. Two of these bind with higher affinity to one of the b 2 sites than to the beta(+) sites. We deduce that 4-benzoyl-propofol binds with.100-fold higher affinity to the gamma+-beta(-)2 site than to the alpha(+)-beta(-) or beta(-) -alpha(-) sites, whereas loreclezole, an anticonvulsant, binds with 5-and 100-fold higher affinity to the alpha(+)-beta(-) site than to the beta(+) and gamma(+) -beta(-) sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABA(A)R transmembrane domain, a property that may facilitate the development of subtype selective GABA(A)R PAMs.