Inhibitable photolabeling by neurosteroid diazirine analog in the β3-Subunit of human hetereopentameric type A GABA receptors

Abstract Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABA A receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric β3 GABA A receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABA A receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABA A receptors. Here, we report on the synthesis and pharmacological characterization of several photoaffinity analogs of pregnanolone and allopregnanolone, of which 21-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human α1β3 and α1β3γ2L GABA A receptors at sub-micromolar concentrations. [ 3 H]21-pTFDBzox-AP photoincorporated in a pharmacologically specific manner into the α and β subunits of those receptors, with the β3 subunit photolabeled most efficiently. Importantly, photolabeling by [ 3 H]21-pTFDBzox-AP was inhibited by the positive steroid modulators alphaxalone, pregnanolone and allopregnanolone, but not by inhibitory neurosteroid pregnenolone sulfate or by two potent general anesthetics and GABA A R positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABA A R that are different from those interacting with neurosteroids. 21-pTFDBzox-AP's potency and pharmacological specificity of photolabeling indicate its suitability for characterizing neurosteroid binding sites in native GABA A receptors.