Optimized expression and purification of NavAb provide the structural insight into the voltage dependence
Optimized expression and purification of NavAb provide the structural insight into the voltage dependence
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DOI：
10.1002/1873-3468.12955
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发表时间：
2018-01-01
期刊：
影响因子：
3.5
通讯作者：
Fujiyoshi, Yoshinori
中科院分区：
文献类型：
--
作者：
Irie, Katsumasa;Haga, Yukari;Fujiyoshi, Yoshinori
电压门控钠通道对于生命系统中的电信号至关重要。分子机制的分析需要精细的电生理评估和高分辨率的通道结构。在此，我们优化了NavAb的双表达系统，该系统是原核电压门控钠通道的成熟标准品，用于E.大肠杆菌和昆虫细胞使用单一质粒载体分析高分辨率蛋白质结构和测量大离子电流。使用该表达系统，我们评估了电压依赖性，并确定了NavAb野生型和两个突变体E32Q和N49K的晶体结构，其电压依赖性正移，并且在电压传感器域中失去了必要的相互作用。结构和功能的比较阐明了原核细胞电压门控钠通道电压依赖性的分子机制。
Voltage-gated sodium channels are crucial for electro-signalling in living systems. Analysis of the molecular mechanism requires both fine electrophysiological evaluation and high-resolution channel structures. Here, we optimized a dual expression system of NavAb, which is a well-established standard of prokaryotic voltage-gated sodium channels, for E. coli and insect cells using a single plasmid vector to analyse high-resolution protein structures and measure large ionic currents. Using this expression system, we evaluated the voltage dependence and determined the crystal structures of NavAb wild-type and two mutants, E32Q and N49K, whose voltage dependence were positively shifted and essential interactions were lost in voltage sensor domain. The structural and functional comparison elucidated the molecular mechanisms of the voltage dependence of prokaryotic voltage-gated sodium channels.