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

Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABAA receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric β3 GABAA receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABAA receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABAA 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 GABAA receptors at sub-micromolar concentrations. [3H]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 [3H]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 GABAAR positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABAAR 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 GABAA receptors.