Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor
Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor
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DOI：
10.1074/jbc.m113.479725
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发表时间：
2013-07-05
影响因子：
4.8
通讯作者：
Cohen, Jonathan B.
中科院分区：
文献类型：
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作者：
Chiara, David C.;Jayakar, Selwyn S.;Cohen, Jonathan B.
GABA A型受体(GABA(A)R)是大脑中主要的抑制性神经递质受体，是许多全身麻醉药的靶标，包括挥发性麻醉药、依托咪酯、异丙酚和巴比妥酸盐。然而，这些结构不同的药物是否可以类似地作为GABA(A)Rs的正变构调节剂仍不清楚。以前，光反应性依托咪酯类似物在β(+)-α(-)亚单位界面的跨膜区域中发现了两个等价的麻醉剂结合部位，这两个部位也包含细胞外区域的GABA结合部位。在这里，我们使用R-[H-3]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)巴比妥酸(R-mTFD-MPAB)，一种有效的立体特异性巴比妥酸盐麻醉剂，光标记表达人α1β3伽玛2GABA(A)Rs。蛋白质显微测序显示，R-[~3H]mTFD-MPAB没有光标记α(+)-β(-)亚单位界面上的依托咪酯位点。相反，它在跨膜结构域的α(+)-β(-)和伽马(+)-β(-)亚单位界面上进行光标记。在(类似)-侧，α1m3标记在Ala-291，Tyr294和Gamma 2M3标记在Ser-301，而在(-)-侧，β3M1标记在Met-227。这些残基，就像依托咪酯位点上的残基一样，位于跨膜区突触侧附近的亚单位界面上。相对于α(+)-β(-)/γ(+)-β(-)界面，R-依托咪酯对β(+)-α(-)界面的选择性是>；100倍，而R-mTFD-MPAB对其部位的选择性是>；50倍。每一种配体都可以增强另一种配体的光结合，显示出两个位点之间的变构相互作用。巴比妥酸盐、依托咪酯和异丙酚衍生物的结构异质性可以通过对这两类位点的不同选择性来调节。我们假设，在这些同源亚基间的任何一个位点上的结合足以发挥麻醉作用，这在某种程度上解释了麻醉药令人费解的结构异质性。
GABA type A receptors (GABA(A)R), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. Howsuch structurally diverse agents can act similarly as positive allosteric modulators of GABA(A) Rs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the beta(+)-alpha(-) subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[H-3]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human alpha 1 beta 3 gamma 2 GABA(A)Rs. Protein microsequencing revealed that R-[3H]mTFD-MPAB did not photolabel the etomidate sites at the alpha(+) -beta(-) subunit interfaces. Instead, it photolabeled sites at the alpha(+) -beta(-) and gamma(+) -beta(-) subunit interfaces in the transmembrane domain. On the (similar to)-side, alpha 1M3 was labeled at Ala-291 and Tyr294 and gamma 2M3 at Ser-301, and on the (-)-side, beta 3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the beta(+) -alpha(-) interface relative to the alpha(+) -beta(-)/gamma(+)-beta(-) interfaces was > 100-fold, whereas that of R-mTFD-MPAB for its sites was > 50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. Wehypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.