Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor

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.