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

Allopregnanolone (3 alpha 5 alpha-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABA(A)receptors (GABA(A)Rs) within vivoanesthetic, 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 alpha 1 beta 3 and alpha 1 beta 3 gamma 2 GABA(A)Rs by photoaffinity labeling with [H-3]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([H-3]21-pTFDBzox-AP), a potent GABA(A)R PAM. Protein microsequencing established 3 alpha 5 alpha-P inhibitable photolabeling of amino acids near the cytoplasmic end of the beta subunit M4 (beta 3Pro-415, beta 3Leu-417, and beta 3Thr-418) and M3 (beta 3Arg-309) helices located at the base of a pocket in the beta(+)-alpha(-)subunit interface that extends to the level of alpha Gln-242, a steroid sensitivity determinant in the alpha M1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3 alpha-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3 alpha-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3 alpha 5 alpha-P, as well as 3 beta-OH analogs that are GABA(A)R antagonists, bound with at least 1000-fold lower affinity than 3 alpha 5 alpha-P. Similarly, for GABA(A)R PAMs with the C-20 carbonyl of 3 alpha 5 alpha-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 beta(+)-alpha(-) subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.