Abstract
NaChBac is a bacterial voltage-gated sodium (Nav) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Nav channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of NavRh, a marine bacterial NaChBac ortholog. Two Na+ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na+ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca2+ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na+ and Ca2+ to the vestibule of the SF. In addition, Na+ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Nav orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na+ over Ca2+ in mammalian Nav channels.
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Acknowledgements
This work was supported by funds from the Ministry of Science and Technology of China (2011CB911102, 2011CB910501 and 2009CB918802), the National Natural Science Foundation of China (31125009 and 91017011) and Tsinghua University. The computational work was also supported by the Tsinghua National Laboratory for Information Science and Technology.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Table S1
Summary of equilibrium simulations (PDF 46 kb)
Supplementary information, Table S2
Statistics of diffraction data collection and structural refinement (PDF 56 kb)
Supplementary information, Table S3
Free energy changes calculated in the FEP method (PDF 64 kb)
Supplementary information, Table S4
The windows and the corresponding simulation steps in FEP calculations (PDF 71 kb)
Supplementary information, Movie S1
Na+ ion permeates NavRh through two successive binding sites. The animation is generated based on simulation 1 in Supplementary information, Table S1, in the presence of 70 mM NaCl. Two diagonal subunits are omitted for visual clarity. The groups in SF are shown in sticks while the pore domain in cartoon. Na+ ions are represented by brown spheres. The same color scheme is applied unless otherwise indicated. (MPEG 7301 kb)
Supplementary information, Movie S2
Ca2+ stably binds at site 1, lacking the ability to pass through the SF. The animation is generated based on simulation 3, in the presence of 70 mM CaCl2. Ca2+ ions are represented by green spheres. (MPEG 7459 kb)
Supplementary information, Movie S3
Ca2+ goes through the SF with low probability. The animation is generated based on simulation 5, in the presence of 200 mM CaCl2. (MPEG 6835 kb)
Supplementary information, Movie S4
Ca2+ blocks Na+ current from the extracellular side by binding Glu183 at site 1. The animation is generated based on simulation 7, in the presence of 70 mM NaCl. A Ca2+ ion was initially restricted near site 1. (MPEG 4101 kb)
Supplementary information, Movie S5
Ca2+ snugly stays at the center of site 2, if pre-restricted in the cavity. The animation is generated based on simulation 8, in the presence of 70 mM CaCl2. A Ca2+ ion was initially restricted in the cavity beneath SF. (MPEG 4431 kb)
Supplementary information, Figure S1
The selectivity filter (SF) of NavRh automatically restores from the collapsed state (crystal structure) and allows the passage of ions during the simulations. (PDF 243 kb)
Supplementary information, Figure S2
Structural changes of site 2 during Na+ permeation. (PDF 304 kb)
Supplementary information, Figure S3
The free-energy profiles estimated from the negative logarithm of Na+ density in the MD simulation of NavRh pore domain as a function of the axial position of SF. (PDF 142 kb)
Supplementary information, Figure S4
Initial arrival of Ca2+ at site 1. (PDF 101 kb)
Supplementary information, Figure S5
The numbers of coordinating atoms around the target ions. (PDF 517 kb)
Supplementary information, Figure S6
The radial distribution function of oxygen atoms donated by water (solid) and protein (dashed) for the target cation. (PDF 183 kb)
Supplementary information, Figure S7
Site 2 is able to accommodate various ions. (PDF 400 kb)
Supplementary information, Figure S8
Na+ causes larger conformational changes of SF than Ca2+ does when binding to site 2. (PDF 269 kb)
Supplementary information, Figure S9
Na+ penetrates NavRh away from the axial center of SF. (PDF 378 kb)
Supplementary information, Figure S10
Ser180 facilitates Na+ transition between site 1 and site 2. (PDF 486 kb)
Supplementary information, Figure S11
Backbone RMSD of the simulated structures against the crystal NavRh structure. (PDF 224 kb)
Supplementary information, Figure S12
The change of system energy in the last 0.5 ns in the pre-equilibrium in all 15 simulations. (PDF 298 kb)
Supplementary information, Figure S13
The schematic representation of the Free Energy Perturbation (FEP) method. (PDF 70 kb)
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Zhang, X., Xia, M., Li, Y. et al. Analysis of the selectivity filter of the voltage-gated sodium channel NavRh. Cell Res 23, 409–422 (2013). https://doi.org/10.1038/cr.2012.173
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DOI: https://doi.org/10.1038/cr.2012.173
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