FIGURE 12.

S-mTFD-MPPB-binding sites at subunit interfaces within the transmembrane domain of an α1β3γ2 GABA_AR. A–F, views are shown of an α1β3γ2 GABA_AR homology model built using the β3 GABA_AR crystal structure (PDB 4COF), with subunits color-coded (α1, light yellow; β3, light blue; and γ2, green), and amino acids of interest shown in stick representation, color-coded to match their colors in the primary structure alignment of the M1-M3 region of the three subunits (G). Views from the side (A) and of the TMD from the ECD-TMD interface (B) include the locations of GABA- (blue) and benzodiazepine (BZD, purple)-binding sites in the ECD and the pharmacologically distinct binding sites in the TMD for S-mTFD-MPPB at the γ⁺-β⁻ (yellow), β⁺-α⁻ (olive), and α⁺-β⁻ (brown) interfaces. Illustrated in C, E and D, F are details of the pockets at γ⁺-β⁻ and β⁺-α⁻ interfaces, respectively, viewed from the lipid (C and D) and from the base of the ECD (E and F). The residues photolabeled by S-[³H]mTFD-MPPB (γ2Ser-280 (γM2–15′), β3Met-227 and β3Leu-231, β3Thr-262, β3Met-286, β3Phe-289, and α1Met-236) are shown in stick representation, as well as two residues (gray) that are not photolabeled as follows: γ2Phe-304, at the γ⁺-α⁻ interface in a position equivalent to β3Phe-289 at the β⁺-α⁻ interface, and β3Asn-265 (βM2–15′) at the β⁺-α⁻ interface, an in vitro and in vivo sensitivity determinant for the GABA_AR potentiating and anesthetic effects of etomidate and propofol (9, 38). Also included in C–F are the Connolly surfaces enclosing the 10 lowest energy docking solutions for S-mTFD-MPPB at each interface pocket. The volume within the Connolly surfaces at the γ⁺-α⁻ and β⁺-α⁻ interfaces are 590 and 420 ų, respectively, and 315 ų for a single molecule. C and E, S-mTFD-MPPB is shown in stick representation at the γ⁺-β⁻ interface in a low energy solution (CDocker interaction energy −38.3 kcal/mol) with the diazirine ∼3 Å from γ2Ser-280 (M2–15′), the GABA_AR amino acid photolabeled most efficiently (+bicuculline). R-[³H]mTFD-MPAB photolabels β3Met-227 but not γ2Ser-280. At the β⁺-α⁻ interface, [³H]azietomidate photolabels β3Met-286 and α1Met-236 (21, 22).