Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor*

GABA type A receptors (GABA A R), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABA A Rs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β + -α − subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R -[ 3 H]5-allyl-1-methyl-5-( m -trifluoromethyl-diazirynylphenyl) barbituric acid ( R-m TFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABA A Rs. Protein microsequencing revealed that R -[ 3 H] m TFD-MPAB did not photolabel the etomidate sites at the β + -α − subunit interfaces. Instead, it photolabeled sites at the α + -β − and γ + -β − subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (−)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R -etomidate for the β + -α − interface relative to the α + -β − /γ + -β − interfaces was >100-fold, whereas that of R-m TFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics. Background: General anesthetics of diverse chemical structure potentiate GABA A receptors by binding to unknown sites. Results: A photoreactive barbiturate identifies intersubunit-binding sites distinct from, but homologous to, sites identified by photoreactive etomidate analogs. Conclusion: Propofol, barbiturates, and etomidate analogs bind with variable selectivities to two classes of sites. Significance: This study helps define the diversity of GABA A receptor general anesthetic-binding sites.