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

GABAA receptors (GABAARs) 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 ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α1β3γ2 GABAAR transmembrane domain at β +-α - (β + sites) and α +-β -/γ +-β - (β - sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-MPAB 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 β +, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to β - sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α +-β - and γ +-β - sites. However, we discovered four compounds that bind with different affinities to the two β - interface sites. Two of these bind with higher affinity to one of the β - sites than to the β + sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ +-β - site than to the α +-β - or β +-α - sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α +-β - site than to the β + and γ +-β - sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.