Figure 3.

The neurosteroid potentiation site. (a) Pregnanolone (ball-and-stick representation, carbon atoms in blue, oxygen atoms in red) bound to an inter-subunit site between M3 residues on the principal face of one subunit (P in box) and M1 residues on the complementary face of the next (C in box). Putative hydrogen bonds between the pregnanolone 3α-hydroxyl and the Gln245 amide (2.4 Å) and C20 ketone and Thr309 hydroxyl (3.0 Å) are indicated by dashed lines. (b) and (c) Modelled heteromeric β_P-α_C and α_P-β_C interfaces respectively, in cartoon and surface representation, in which the relevant α5_TMD face is substituted by the equivalent β3 subunit structure (PDB ID: 4COF). Note that α5 residues in these two panels are labelled by letters only, without numbers, to reflect their conservation across the α subunits (α1-6) within this site. Pregnanolone is well accommodated in the heteromeric β_P-α_C site (b) but not in the α_C-β_P site (c) due to the replacement of α M1 Gln with the β3 M1 Trp237, which closes the top of the pocket (dashed line). (d) Sequence alignment of the M1 and M3 motifs containing the neurosteroid binding site residues (bold) in the GABA_AR α5 subunit and equivalent regions in other human pLGIC superfamily members. M1 Gln245, unique to GABA_AR α-subunits, is highlighted in red. GABA_AR β-subunit residues contributed from the complementary face in heteromeric receptors are boxed. (e) Pregnanolone and (f) Allopregnanolone dose-response curves for potentiation of GABA EC_10-15 responses. Alanine substitution of β3 principal face residues Leu297, but not Phe301, reduces neurosteroid sensitivity. These residues are highlighted by black boxes in b. Each data point represents mean ± s.e.m. Pregnanolone EC₅₀ n number: EC₅₀: WT n = 13, L297A n = 11, F301A n = 8. Allopregnanolone EC₅₀: WT n = 5, L297A n = 9, F301A n = 4. Each n EC₅₀ measurement is from different cells.