Binding mechanism analysis between DIVS6 model peptides of voltage-gated sodium channel and a lipid soluble alkaloid veratridine using solution NMR
Binding mechanism analysis between DIVS6 model peptides of voltage-gated sodium channel and a lipid soluble alkaloid veratridine using solution NMR
复制标题
使用溶液NMR分析电压门控钠通道DIVS6模型肽与脂溶性生物碱藜芦碱的结合机制
DOI：
10.1016/j.bmc.2012.03.034
复制
发表时间：
2012
影响因子：
3.5
通讯作者：
Kazuo Tachibana
中科院分区：
文献类型：
--
作者：
Ai Yoshinaka-Niitsu;Tohru Yamagaki;Masanori Harada;Kazuo Tachibana
电压门控钠通道（VGSC）负责在神经系统中产生动作电位。藜芦定（Veratridine，VTD）是一种脂溶性生物碱，可与VGSC的第6节结合，具有部分激动作用。然而，由于VGSC的大尺寸和膜定位，VGSC和VTD之间的高分辨率结构相互作用机制难以阐明。在此，作者设计了对应于大鼠骨骼肌Nav1.4的结构域IV片段6（DIVS 6）的模型肽，并通过溶液NMR分析来分析模型肽与VTD的复合物，以获得相互作用的结构信息。模型肽在膜模拟溶剂2，2，2-三氟乙醇水溶液中成功形成了α-螺旋，这是DIVS 6的疑似天然构象。利用VTD的NMR滴定实验鉴定了模型肽的VTD结合残基，包括一个新发现的VTD结合残基Leu 14（Nav1.4中的μ1-L1580），该残基尚未通过点突变研究报道。模型肽上的VTD结合残基的映射揭示了疏水相互作用表面。用VTD的无毒类似物维拉卡因的NMR滴定实验也表明VTD的甾体骨架与DIVS 6的疏水相互作用表面相互作用，并且VTD的3-酰基可能通过与结构域I片段6和/或结构域IV片段4相互作用而引起神经毒性。
Voltage-gated sodium channels (VGSCs) are responsible for generating action potentials in nervous systems. Veratridine (VTD), a lipid soluble alkaloid isolated from sabadilla lily seed, is believed to bind to segment 6 of VGSCs and act as a partial agonist. However, high resolution structural interaction mechanism between VGSCs and VTD is difficult to elucidate because of the large size and membrane localization of VGSCs. Here, the authors designed model peptides corresponding to domain IV segment 6 (DIVS6) of rat skeletal muscle Nav1.4 and analyzed the complex of the model peptides and VTD by solution NMR analysis to obtain structural information of the interaction. The model peptides successfully formed an α-helices, which is the suspected native conformation of DIVS6, in aqueous 2,2,2-trifluoroethanol, a membrane-mimicking solvent. The VTD binding residues of the model peptide were identified using the NMR titration experiments with VTD, including a newly discovered VTD binding residue Leu14 (μ1-L1580 in Nav1.4), which has not been reported by point mutation studies. Mapping of VTD binding residues on the model peptide revealed the hydrophobic interaction surface. NMR titration experiments with a non-toxic analog of VTD, veracevine, also indicated that the steroidal backbone of VTD interacts with the hydrophobic interaction surface of DIVS6 and that the 3-acyl group of VTD possibly causes neurotoxicity by interacting with domain I segment 6 and/or domain IV segment 4.