Allopregnanolone (3α5α-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 α1β3 and α1β3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM.
