Tao Feng, Chen Qiang, Sato Yuko, Skinner John, Tang Pei, Johns Roger A
From the Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland (F.T., Y.S., J.S., R.A.J.); Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, Texas (F.T.); and Departments of Anesthesiology (Q.C., P.T.), Pharmacology and Chemical Biology (P.T.), and Computational Biology (P.T.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Anesthesiology. 2015 Apr;122(4):776-86. doi: 10.1097/ALN.0000000000000609.
The authors have shown previously that inhaled anesthetics disrupt the interaction between the second postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 (PDZ) domain of postsynaptic density protein-95 (PSD-95) and the C-terminus of N-methyl-D-aspartate receptor subunits NR2A and NR2B. The study data indicate that PDZ domains may serve as a molecular target for inhaled anesthetics. However, the underlying molecular mechanisms remain to be illustrated.
Glutathione S-transferase pull-down assay, coimmunoprecipitation, and yeast two-hybrid analysis were used to assess PDZ domain-mediated protein-protein interactions in different conditions. Nuclear magnetic resonance spectroscopy was used to investigate isoflurane-induced chemical shift changes in the PDZ1-3 domains of PSD-95. A surface plasmon resonance-based BIAcore (Sweden) assay was used to examine the ability of isoflurane to inhibit the PDZ domain-mediated protein-protein interactions in real time.
Halothane and isoflurane dose-dependently inhibited PDZ domain-mediated interactions between PSD-95 and Shaker-type potassium channel Kv1.4 and between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA2 and its interacting proteins-glutamate receptor-interacting protein or protein interacting with c kinase 1. However, halothane and isoflurane had no effect on PDZ domain-mediated interactions between γ-aminobutyric acid type B receptor and its interacting proteins. The inhaled anesthetic isoflurane mostly affected the residues close to or in the peptide-binding groove of PSD-95 PDZ1 and PDZ2 (especially PDZ2), while barely affecting the peptide-binding groove of PSD-95 PDZ3.
These results suggest that inhaled anesthetics interfere with PDZ domain-mediated protein-protein interactions at several receptors important to neuronal excitation, anesthesia, and pain processing.
作者先前已表明，吸入麻醉药会破坏突触后致密蛋白-95（PSD-95）的第二个突触后致密蛋白-95、果蝇盘大肿瘤抑制因子和紧密连接蛋白-1（PDZ）结构域与N-甲基-D-天冬氨酸受体亚基NR2A和NR2B的C末端之间的相互作用。研究数据表明，PDZ结构域可能是吸入麻醉药的分子靶点。然而，其潜在的分子机制仍有待阐明。
采用谷胱甘肽S-转移酶下拉试验、免疫共沉淀和酵母双杂交分析来评估不同条件下PDZ结构域介导的蛋白质-蛋白质相互作用。利用核磁共振光谱研究异氟烷诱导的PSD-95的PDZ1-3结构域的化学位移变化。采用基于表面等离子体共振的BIAcore（瑞典）试验实时检测异氟烷抑制PDZ结构域介导的蛋白质-蛋白质相互作用的能力。
氟烷和异氟烷剂量依赖性地抑制PSD-95与震颤型钾通道Kv1.4之间以及α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体亚基GluA2与其相互作用蛋白——谷氨酸受体相互作用蛋白或与c激酶1相互作用的蛋白之间的PDZ结构域介导的相互作用。然而，氟烷和异氟烷对γ-氨基丁酸B型受体与其相互作用蛋白之间的PDZ结构域介导的相互作用没有影响。吸入麻醉药异氟烷主要影响PSD-95的PDZ1和PDZ2（尤其是PDZ2）的肽结合槽附近或其中的残基，而对PSD-95的PDZ3的肽结合槽影响很小。
这些结果表明，吸入麻醉药在对神经元兴奋、麻醉和疼痛处理重要的几种受体处干扰PDZ结构域介导的蛋白质-蛋白质相互作用。
