Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α 1 β 3 γ 2 GABAAR Transmembrane Domain

Abstract

GABA_A receptors (GABA_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 ([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 GABA_AR 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 GABA_AR transmembrane domain, a property that may facilitate the development of subtype selective GABA_AR PAMs.