文章基本信息

标题：The activated state of a sodium channel voltage sensor in a membrane environment
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作者：Sudha Chakrapani ; Pornthep Sompornpisut ; Pathumwadee Intharathep 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2010
卷号：107
期号：12
页码：5435-5440
DOI：10.1073/pnas.0914109107
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
关键词：complementation assays ; EPR spectroscopy ; gating charges ; electrical excitability