TY - JOUR T1 - Neurosteroid Analogue Photolabeling of a Site in the TM3 Domain of the β<sub>3</sub> Subunit of the GABA<sub>A</sub> Receptor JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.112.078410 SP - mol.112.078410 AU - Zi-Wei Chen AU - Brad Manion AU - R Reid Townsend AU - Douglas F Covey AU - David E Reichert AU - Joe Henry Steinbach AU - Werner Sieghart AU - Karoline Fuchs AU - Alex S Evers Y1 - 2012/05/30 UR - http://molpharm.aspetjournals.org/content/early/2012/05/30/mol.112.078410.abstract N2 - Accumulated evidence suggests that neurosteroids modulate GABAA receptors via binding interactions with their transmembrane domains. To directly identify these neurosteroid binding sites, a neurosteroid analogue photolabeling reagent, (3α,5β)-6-Azi-pregnanolone (6-AziP), was used to photolabel membranes from sf-9 cells expressing high density recombinant His8-β3 homomeric GABAA receptors. 6-AziP inhibited [35S]t-butylbicyclophosphorothionate binding to the His8-β3 homomeric GABAA receptors in a concentration-dependent manner (IC50 = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [3H]6-AziP photolabeled proteins of 30, 55, 110 and 150 kDa in a concentration-dependent manner. The 55, 110 and 150 kDa proteins were identified as His8-β3 subunits by immunoblot and by enrichment on a Ni-affinity column. Photolabeling of the β3 subunits was stereoselective, with [3H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [3H](3β,5β)-6-AziP. High resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His8-β3 subunits identified a single photolabeled peptide, ALLEYAF6-AziP, in the third transmembrane domain (TM3). The identity of this peptide and the site of incorporation on Phe301 were confirmed by high-resolution tandem mass spectra (MS2). No other sites of photoincorporation were observed despite obtaining 90% sequence coverage of the whole β3 subunit protein including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites using mass spectrometry. ER -