Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC
Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC
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DOI：
10.1016/j.bpj.2010.07.023
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发表时间：
2010-09-22
影响因子：
3.4
通讯作者：
Tang, Pei
中科院分区：
文献类型：
--
作者：
Chen, Qiang;Cheng, Mary Hongying;Tang, Pei
半胱氨酸环受体是全身麻醉药的分子靶点，但麻醉剂与这些蛋白质结合的知识仍然有限。在这里，我们调查麻醉剂结合细菌Gloeulopsis violaceus五聚体配体门控离子通道（GLIC），一个结构同源的cys环受体，使用实验和计算的混合方法。色氨酸荧光猝灭实验表明，氟烷和硫喷妥钠结合在三个与色氨酸相关的位点，在细胞外（EC）结构域，跨膜（TM）结构域，和EC-TM界面的GLIC。通过对接分析预测了EC-TM界面处的额外结合位点，并通过对N200 W GLIC突变体的淬灭实验进行了验证。分别从氟烷和硫喷妥钠的荧光猝灭数据推导出2.3 +/- 0.1 mM和0.10 +/- 0.01 mM的结合亲和力（K-D）。对接这些麻醉剂的原始GLIC晶体结构和结构放松的分子动力学模拟揭示intrasubunit网站大多数氟烷结合和intersubunit网站硫喷妥钠结合。色氨酸的范围内和亚基间的结合位点。在不同位点存在氟烷的情况下对GLIC进行的多个分子动力学模拟表明，EC-TM界面处的麻醉剂结合破坏了通道门控的关键相互作用，改变了TM 23接头的运动，并使开放通道构象不稳定，这可能导致GLIC通道电流的抑制。该研究不仅提供了对GLIC中麻醉剂结合的见解，而且还展示了实验和计算的成功融合，以了解复杂蛋白质中的麻醉剂作用。
Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K-D) of 2.3 +/- 0.1 mM and 0.10 +/- 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.