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bioRxiv · 10.1101/2023.04.24.538027

Determining The Structure of the Bacterial Voltage-gated Sodium Channel NaChBac Embedded in Liposomes by Cryo Electron Tomography and Subtomogram Averaging

Chang, S.-Y. S.·Dijkman, P. M.·Wiessing, S. A.·Kudryashev, M.
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Abstract

Voltage-gated sodium channels shape action potentials that propagate signals along cells. When the membrane potential reaches a certain threshold, the channels open and allow sodium ions to flow through the membrane depolarizing it, followed by the deactivation of the channels. Opening and closing of the channels is important for cellular signalling and regulates various physiological processes in muscles, heart and brain. Mechanistic insights into the voltage-gated channels are difficult to achieve as the proteins are typically extracted from membranes for structural analysis which results in the loss of the transmembrane potential. Here, we report the structural analysis of a bacterial voltage-gated sodium channel, NaChBac, reconstituted in liposomes under an electrochemical gradient by cryo electron tomography and subtomogram averaging. We show that the small channel, most of the residues of which are embedded in a membrane, can be localized using a genetically fused GFP. GFP can aid the initial alignment to an average resulting in a correct structure, but does not help for the final refinement. At a moderate resolution of [~]16 [A] the structure of NaChBac in an unrestricted membrane bilayer is 10% wider than the structure of a purified protein previously solved in nanodiscs, suggesting the potential movement of the peripheral voltage-sensing domains. Our structural analysis explores the limits of structural analysis of membrane proteins in membranes. HIGHLIGHTSO_LIStructural analysis of the bacterial voltage-gated sodium channel NaChBac in lipid vesicles under the resting membrane potential by cryo electron tomography and subtomogram averaging. C_LIO_LIFused GFP allows identification of a 120-kDa mostly transmembrane protein in tomograms, and helps for the initial alignment but not for the final refinements. C_LIO_LIThe map of NaChBac in liposomes at a resolution of 16.3 [A] is [~]10% wider than the protein structure in a nanodisc. C_LI

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Chang, S.-Y. S., Dijkman, P. M., Wiessing, S. A., Kudryashev, M.. 2023-04-24. Determining The Structure of the Bacterial Voltage-gated Sodium Channel NaChBac Embedded in Liposomes by Cryo Electron Tomography and Subtomogram Averaging. https://doi.org/10.1101/2023.04.24.538027

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