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

Abstract

Allopregnanolone (3a5a-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) 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 GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human a1b3 and a1b3g2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl) benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3a5a-P inhibitable photolabeling of amino acids near the cytoplasmic end of the b subunit M4 (b3Pro-415, b3Leu-417, and b3Thr-418) and M3 (b3Arg-309) helices located at the base of a pocket in the b1-a2 subunit interface that extends to the level of aGln-242, a steroid sensitivity determinant in the aM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3a-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3a-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3a5a-P, as well as 3b-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3a5a-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3a5a-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 GABAAR b1-a2 subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.