Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the beta 3 Subunit of the GABA(A) Receptor

作者：Chen Zi Wei; Manion Brad; Townsend R Reid; Reichert David E; Covey Douglas F; Steinbach Joe Henry; Sieghart Werner; Fuchs Karoline; Evers Alex S^*

来源：Molecular Pharmacology, 2012, 82(3): 408-419.

DOI：10.1124/mol.112.078410

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3 beta,5 beta)-6-azipregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-beta 3 homomeric GABA(A) receptors. 6-AziP inhibited S-35-labeled t-butylbicy-clophosphorothionate binding to the His(8)-beta 3 homomeric GABA(A) receptors in a concentration-dependent manner (IC50 = 9 +/- 1 mu M), with a pattern consistent with a single class of neurosteroid binding sites. [H-3] 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 His(8)-beta 3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the beta 3 subunits was stereoselective, with [3H] 6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [H-3](3 beta,5 beta)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziPlabeled His(8)-beta 3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole beta 3 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 by using mass spectrometry.

• 出版日期2012-9