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首页> 外文期刊>Biochemistry >Mapping General Anesthetic Binding Site(s) in Human α1β3 γ-Aminobutyric Acid Type A Receptors with [~3H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue
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Mapping General Anesthetic Binding Site(s) in Human α1β3 γ-Aminobutyric Acid Type A Receptors with [~3H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue

机译：用[〜3H] TDBzl-依托咪酯（一种光反应性依托咪酯类似物）在人α1β3γ-氨基丁酸A型受体中定位全身麻醉剂的结合位点

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The γ-aminobutyric acid type A receptor (GABA_AR) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA_ARs purified from bovine brain, [~3H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABAAR transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599?11605]. To further define GABAAR etomidate binding sites, we now use [3H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA_ARs and more extensively identify photolabeled GABA_AR amino acids. [~3H]TDBzletomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [~3H]azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA_AR homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [~3H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA_AR intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [~3H]TDBzl-etomidate and [~3H]azietomidate also provides evidence of a homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABA_AR.
机译：γ-氨基丁酸A型受体（GABA_AR）是多种化学结构的全身麻醉剂的靶标，在临床剂量下它们可作为正变构调节剂。以前，在从牛脑中纯化的GABA_ARs的异质混合物中，αM1和βM3跨膜螺旋中[〜3H]叠氮嘧啶光标记αMet-236和βMet-286的分子在GABAAR跨膜结构域中的β之间的界面处确定了一个依托咪酯结合位点和α亚基[Li，GD，等。 （2006）J. Neurosci。 26，11599？11605]。为了进一步定义GABAAR依托咪酯的结合位点，我们现在使用[3H] TDBzl-依托咪酯（一种比二氮丙啶具有更宽的氨基酸侧链反应性的芳基重氮）来光标记纯化的人FLAG-α1β3GABA_ARs，并更广泛地鉴定光标记的GABA_AR氨基酸。 [〜3H] TDBzletomidate以依托咪酯抑制方式在β3M3跨膜螺旋中以及在α1M1中的α1Met-236（以[〜3H] azietomidate进行光标记的残基）上进行光标记，而在αM2中没有对氨基酸进行光标记。检测到也与依托咪酯结合位点接壤的βM2螺旋。这些光标记的氨基酸在GABA_AR同源性模型中的位置，该模型来自最近确定的原核生物（GLIC）或无脊椎动物（GluCl）同源物结构，计算机对接研究的结果预测[〜3H] TDBzl-依托咪酯在该位点的结合方向以及其他有助于该GABA_AR亚单位依托咪酯结合位点的氨基酸。 [〜3H] TDBzl-etomidate和[〜3H] azietomidate抑制依托咪酯对βM1中的β3Met-227的光标记，也提供了在α1β3GABA_AR的β3-β3亚基界面上有一个依托咪酯结合位点的证据。

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