The activated state of a sodium channel voltage sensor in a membrane environment
The activated state of a sodium channel voltage sensor in a membrane environment
复制标题
DOI：
10.1073/pnas.0914109107
复制
发表时间：
2010-03-23
影响因子：
11.1
通讯作者：
Perozo, Eduardo
中科院分区：
文献类型：
--
作者：
Chakrapani, Sudha;Sompornpisut, Pornthep;Perozo, Eduardo
渗透，选择性和门控的基本机制的直接结构的见解仍然不可用的Na+和Ca 2+通道的家庭。在这里，我们报告的分离的电压传感器域（VSD）的原核Na+通道NaChBac在脂质双层的光谱结构表征。定点自旋标记和EPR光谱进行了118个突变体，涵盖所有的VSD。EPR环境数据被用来明确分配的二级结构元素，定义膜插入限制，并评估激活构象的隔离VSD在膜中使用约束驱动的分子动力学模拟。NaChBac-VSD的整体三维折叠与KvAP、Kv1.2、Kv1.2-2.1嵌合体和MlotiK 1中观察到的折叠非常相似。然而，与膜包埋的KvAP-VSD相比，NaChBac-VSD的结构动力学揭示了更紧密的螺旋包装，在门控电荷的局部环境及其与蛋白质其余部分的相互作用中存在细微差异。使用细胞互补分析，我们表明，NaChBac-VSD可以提供一个管道，以运输的离子在休息或“向下”的构象，一个功能与我们的EPR水的可及性测量在激活或“向上”的构象一致。这些结果表明，VSD的整体架构是显着保守的K+和Na+通道和门控孔电流的途径可能是固有的大多数电压传感器。细胞互补试验还提供了关于“向下/静止”状态下门控电荷的推定位置的信息，因此可以一瞥激活过程中构象变化的程度。
Direct structural insights on the fundamental mechanisms of permeation, selectivity, and gating remain unavailable for the Na+ and Ca2+ channel families. Here, we report the spectroscopic structural characterization of the isolated Voltage-Sensor Domain (VSD) of the prokaryotic Na+ channel NaChBac in a lipid bilayer. Site-directed spin-labeling and EPR spectroscopy were carried out for 118 mutants covering all of the VSD. EPR environmental data were used to unambiguously assign the secondary structure elements, define membrane insertion limits, and evaluate the activated conformation of the isolated-VSD in the membrane using restrain-driven molecular dynamics simulations. The overall three-dimensional fold of the NaChBac-VSD closely mirrors those seen in KvAP, Kv1.2, Kv1.2-2.1 chimera, and MlotiK1. However, in comparison to the membrane-embedded KvAP-VSD, the structural dynamics of the NaChBac-VSD reveals a much tighter helix packing, with subtle differences in the local environment of the gating charges and their interaction with the rest of the protein. Using cell complementation assays we show that the NaChBac-VSD can provide a conduit to the transport of ions in the resting or "down" conformation, a feature consistent with our EPR water accessibility measurements in the activated or "up" conformation. These results suggest that the overall architecture of VSD's is remarkably conserved among K+ and Na+ channels and that pathways for gating-pore currents may be intrinsic to most voltage-sensors. Cell complementation assays also provide information about the putative location of the gating charges in the "down/resting" state and hence a glimpse of the extent of conformational changes during activation.