Department of Biochemistry and Molecular Biology, University of Chicago, Center for Integrative Science, 929 East 57th Street, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2010 Mar 23;107(12):5435-40. doi: 10.1073/pnas.0914109107. Epub 2010 Mar 5.
Direct structural insights on the fundamental mechanisms of permeation, selectivity, and gating remain unavailable for the Na(+) and Ca(2+) 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.
直接的结构见解关于渗透、选择性和门控的基本机制仍然无法获得的 Na(+) 和 Ca(2+) 通道家族。在这里，我们报告了在脂质双层中分离的细菌 Na(+) 通道 NaChBac 的电压传感器结构域 (VSD) 的光谱结构特征。进行了定点自旋标记和 EPR 光谱学研究，涵盖了 VSD 的所有 118 个突变体。EPR 环境数据用于明确分配二级结构元素、定义膜插入限制，并使用约束驱动的分子动力学模拟评估膜中分离-VSD 的激活构象。NaChBac-VSD 的整体三维折叠与 KvAP、Kv1.2、Kv1.2-2.1 嵌合体和 MlotiK1 中观察到的折叠非常相似。然而，与嵌入膜中的 KvAP-VSD 相比，NaChBac-VSD 的结构动力学显示出更紧密的螺旋包装，门控电荷的局部环境及其与蛋白质其余部分的相互作用存在细微差异。使用细胞互补测定法，我们表明 NaChBac-VSD 可以为离子在静止或“向下”构象下的运输提供通道，这一特征与我们在激活或“向上”构象下进行的 EPR 水可及性测量一致。这些结果表明，VSD 的整体架构在 K(+) 和 Na(+) 通道中非常保守，并且门控孔电流的途径可能是大多数电压传感器的固有特性。细胞互补测定法还提供了关于“向下/静止”状态下门控电荷的假定位置的信息，从而可以一窥激活过程中的构象变化程度。
