Li Guo-Dong, Chiara David C, Cohen Jonathan B, Olsen Richard W
Department of Molecular and Medical Pharmacology, Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
J Biol Chem. 2009 May 1;284(18):11771-5. doi: 10.1074/jbc.C900016200. Epub 2009 Mar 12.
Photoaffinity labeling of gamma-aminobutyric acid type A (GABA(A))-receptors (GABA(A)R) with an etomidate analog and mutational analyses of direct activation of GABA(A)R by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABA(A)Rs in the transmembrane domain at the interface between the beta and alpha subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599-11605) azietomidate photolabeling of GABA(A)R alpha1Met-236 and betaMet-286 (in alphaM1 and betaM3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of beta and alpha subunits (two copies per pentamer) is consistent with a GABA(A)R homology model based upon the structure of the nicotinic acetylcholine receptor and with recent alphaM1 to betaM3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA(A)R subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA(A)R-modulating neurosteroids do not inhibit photolabeling of GABA(A)R alpha1Met-236 or betaMet-286 but enhance labeling of alpha1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486-489) are located at the subunit interface defined by our etomidate site model.
用依托咪酯类似物对γ-氨基丁酸A型（GABA(A)）受体（GABA(A)R）进行光亲和标记以及对神经甾体直接激活GABA(A)R的突变分析，均提出这些结构不同的全身麻醉药结合于GABA(A)R跨膜结构域中β和α亚基之间界面处的位点。我们通过检查神经活性甾体是否抑制依托咪酯类似物的光标记，来测试这两个配体结合位点是否可能重叠。我们之前已鉴定出（Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599 - 11605）GABA(A)R α1Met - 236和βMet - 286（在αM1和βM3中）的氮杂依托咪酯光标记。将这两个光标记的氨基酸定位在β和α亚基界面处的单一类型结合位点（每个五聚体有两个拷贝），与基于烟碱型乙酰胆碱受体结构的GABA(A)R同源模型以及最近的αM1至βM3交联数据一致。在分析SDS - PAGE上的GABA(A)R亚基水平进行测定时，生物活性神经甾体增强而非抑制氮杂依托咪酯的光标记，并且蛋白质微量测序确定调节GABA(A)R的神经甾体不抑制GABA(A)R α1Met - 236或βMet - 286的光标记，而是增强α1Met - 236的标记。因此，调节性甾体与依托咪酯不在同一位点结合，并且被鉴定为神经甾体激活决定因素的氨基酸（Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486 - 489）均不在我们的依托咪酯位点模型所定义的亚基界面处。
