摘要：

Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxi-olytic 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 beta 3 GABA(A) receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABA(A) receptor alpha subunit TMD5 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 alpha 1 beta 3 and alpha 1 beta 3 gamma 2L GABA(A) receptors at sub-micromolar concentrations. [H-3]21-pTFDBzox-AP photoincorporated in a pharma-cologically specific manner into the alpha and beta subunits of those receptors, with the beta 3 subunit photo-labeled most efficiently. Importantly, photolabeling by [H-3]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. (C) 2018 Elsevier Masson SAS. All rights reserved.

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