Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor
Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor
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DOI：
10.1124/mol.112.078410
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发表时间：
2012-09-01
影响因子：
3.6
通讯作者：
Evers, Alex S.
中科院分区：
文献类型：
--
作者：
Chen, Zi-Wei;Manion, Brad;Evers, Alex S.
越来越多的证据表明，神经类固醇通过与跨膜结构域的结合相互作用来调节GABA(A)受体。为了直接识别这些神经类固醇结合位点，使用神经类固醇类似物光标记试剂(3 β,5 β)-6-氮孕酮（6-AziP）对表达高密度重组His(8)- β 3同源GABA(a)受体的Sf9细胞膜进行光标记。6-AziP以浓度依赖性的方式抑制s -35标记的t-丁基基-氯硫代酸与His(8)- β 3同质GABA(A)受体的结合（IC50 = 9 +/- 1 μ M），其模式与一类神经类固醇结合位点一致。[H-3] 6-AziP以浓度依赖性方式光标记30、55、110和150 kDa的蛋白。55-、110-和150-kDa蛋白通过免疫印迹和镍亲和柱富集鉴定为His(8)- β 3亚基。β 3亚基的光标记具有立体选择性，与相同浓度的非对映体[H-3](3 β,5 β)-6-AziP相比，[3H] 6-AziP产生了更大的标记。高分辨率质谱分析亲和纯化的6- aziplar标记的His(8)- β 3亚基在第三跨膜结构域鉴定出一个单一的光标记肽ALLEYAF-6-AziP。通过高分辨率串联质谱法确定了该肽的身份及其在Phe301上的结合位点。尽管整个β 3亚基蛋白的序列覆盖率为90%，包括84%的跨膜结构域，但未观察到其他光结合位点。本研究鉴定了一种新的神经类固醇结合位点，并证明了用质谱法鉴定神经类固醇光标记位点的可行性。
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.