Activation mechanisms of voltage-gated K+ and Na+ channels
Final Report Abstract
The project "Activation mechanisms of voltage-gated K+ and Na+ channels" aimed to study the molecular basis for the propagation of action potentials, which is a key mechanism of signal transduction in the human body. To get there, we aimed to determine the structures of model sodium and potassium channels under physiological-like conditions of applied voltage across the membranes. Structures in these so-called "resting states" would provide detailed understanding of how the transmembrane voltage regulates opening and closing of these ion channels. Such understanding could be highly important for highly specific molecular medicine against multiple cardiac and neuronal disorders. Cryo electron microscopy (cryo-EM) and tomography (cryo-ET) which have been revolutionized over the last decade and acknowledged by the Nobel Prize in Chemistry in 2017 were selected as the methods of choice for structural determination. The project started in February 2018 at the Max Planck Institute for Biophysics in Frankfurt on Main. The project team successfully expressed, purified and reconstituted the model bacterial sodium channel NaChBac in proteoliposomes with the applied voltage across the membranes. Cryo-ET datasets were recorded, and subtomogram averaging (StA) was used for structural determination resulting in a structure at a modest resolution of ˜16 Å. The structure showed the overall correct structure with the tetrameric appearance of a pore domain and four voltage-sensing domains. Comparison with a previously reported structure of NaChBac in lipid nanodiscs showed overall similarity, but the StA structure was wider. Further improvements in resolution would be needed to gain mechanistic insights into the activation mechanisms, however this part of the study explored the limits of the current technology. The structure of mammalian potassium channels under voltage was approached using the same set of methods. As membrane protein biochemistry with mammalian membrane multi-protein complexes is significantly more challenging, we spent significant time optimizing the steps of protein expression, purification and stable reconstitution into lipid vesicles. The experience from the NaChBac part of the project makes us confident that with some optimizations of the sample, structural determination will be possible, the group currently continues the project. The grant's second objective was to optimize the process of structural determination of membrane proteins using cryo-ET. For this we developed a workflow called Basics for Electron tomography and Automatic Reconstructions (tomoBEAR). TomoBEAR automated data processing for ion channels, significantly reducing processing time and producing high-resolution structures. Generation of high-quality tomograms is greatly accelerated and there are some options for particle identification. Benchmarking showed that a combination of software packages Dynamo and Relion is effective for membrane protein classification and high-resolution reconstructions. TomoBEAR was used to produce structures of an ion channel RyR1 in native membranes at resolutions of up to 4.0 Å in one month. We believe that with some adjustments and higher throughput it can be possible to obtain structures at resolution of 3 Å which will allow resolving small molecules and lipids in the maps. TomoBEAR also enabled screening of experimental conditions for cryo-ET analysis of ion channels in microvesicles produced by mammalian cells. The workflow tomoBEAR is open-source and is publicly available. Overall the project has significantly advanced our knowledge of ion channel structure and supported the development of the software that greatly accelerates the structural determination process.
Publications
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Subtomogram averaging from cryo-electron tomograms. Methods in Cell Biology, 217-259. Elsevier.
Leigh, Kendra E.; Navarro, Paula P.; Scaramuzza, Stefano; Chen, Wenbo; Zhang, Yingyi; Castaño-Díez, Daniel & Kudryashev, Misha
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Structure of RyR1 in native membranes. EMBO reports, 21(5).
Chen, Wenbo & Kudryashev, Mikhail
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Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM. Nature Communications, 11(1).
Sanchez, Ricardo M.; Zhang, Yingyi; Chen, Wenbo; Dietrich, Lea & Kudryashev, Mikhail
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KAHRP dynamically relocalizes to remodeled actin junctions and associates with knob spirals in Plasmodium falciparum‐infected erythrocytes. Molecular Microbiology, 117(2), 274-292.
Sanchez, Cecilia P.; Patra, Pintu; Chang, Shih‐Ying Scott; Karathanasis, Christos; Hanebutte, Lukas; Kilian, Nicole; Cyrklaff, Marek; Heilemann, Mike; Schwarz, Ulrich S.; Kudryashev, Mikhail & Lanzer, Michael
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Determining the structure of the bacterial voltage-gated sodium channel NaChBac embedded in liposomes by cryo electron tomography and subtomogram averaging. Scientific Reports, 13(1).
Chang, Shih-Ying Scott; Dijkman, Patricia M.; Wiessing, Simon A. & Kudryashev, Misha
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Streamlined structure determination by cryo-electron tomography and subtomogram averaging using TomoBEAR. Nature Communications, 14(1).
Balyschew, Nikita; Yushkevich, Artsemi; Mikirtumov, Vasilii; Sanchez, Ricardo M.; Sprink, Thiemo & Kudryashev, Mikhail
