Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABA A receptors (GABA A Rs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABA A R transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABA A Rs by photoaffinity labeling with [ 3 H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([ 3 H]21- p TFDBzox-AP), a potent GABA A R PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β + –α − subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABA A R antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABA A R PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABA A R β + –α − subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.