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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Pharmacol. | doi: 10.3389/fphar.2019.00953

Cell-Free Expression of Sodium Channel Domains for Pharmacology Studies. Noncanonical Spider Toxin Binding Site in the Second Voltage-Sensing Domain of Human NaV1.4 Channel

 Mikhail Y. Myshkin1,  Roope Mannikko2,  Olesya A. Krumkacheva3, Dmitrii S. Kulbatskii1, Anton O. Chugunov1, 4, 5,  Antonina A. Berkut1, Alexander S. Paramonov1, Mikhail A. Shulepko1, Matvey V. Fedin3, Michael G. Hanna2,  Dimitri M. Kullmann6, Elena G. Bagryanskaya7, Alexander S. Arseniev1, 4, Mikhail P. Kirpichnikov1, 8,  Ekaterina N. Lyukmanova1, 4,  Alexander A. Vassilevski1, 4 and  Zakhar O. Shenkarev1, 4*
  • 1Institute of Bioorganic Chemistry (RAS), Russia
  • 2Department of Molecular Neuroscience, Institute of Neurology, University College London, United Kingdom
  • 3International Tomography Center (RAS), Russia
  • 4Moscow Institute of Physics and Technology, Russia
  • 5National Research University Higher School of Economics, Russia
  • 6Department of Clinical and Experimental Epilepsy, Institute of Neurology, Faculty of Brain Sciences, University College London, United Kingdom
  • 7Novosibirsk Institute of Organic Chemistry (RAS), Russia
  • 8Lomonosov Moscow State University, Russia

Voltage-gated sodium (NaV) channels are essential for the normal functioning of cardiovascular, muscular, and nervous systems. These channels have modular organization; the central pore domain allows current flow and provides ion selectivity, whereas four peripherally located voltage-sensing domains (VSDs-I/IV) are needed for voltage-dependent gating. Mutations in the S4 voltage-sensing segments of VSDs in the skeletal muscle channel NaV1.4 trigger leak (gating pore) currents and cause hypokalemic and normokalemic periodic paralyses. Previously, we have shown that the gating modifier toxin Hm-3 from the crab spider Heriaeus melloteei binds to the S3-S4 extracellular loop in VSD-I of NaV1.4 channel and inhibits gating pore currents through the channel with mutations in VSD-I. Here, we report that Hm-3 also inhibits gating pore currents through the same channel with the R675G mutation in VSD-II. To investigate the molecular basis of Hm-3 interaction with VSD-II, we produced the corresponding 554-696 fragment of NaV1.4 in a continuous exchange cell-free expression system based on the Escherichia coli S30 extract. We then performed a combined NMR and EPR spectroscopy study of isolated VSD-II in zwitterionic DPC/LDAO or DPC micelles. To speed-up the assignment of backbone resonances, five selectively 13C,15N-labeled VSD-II samples were produced in accordance with specially calculated combinatorial scheme. This labeling approach provides assignment for ~ 50% of the backbone. Obtained NMR and EPR data revealed correct secondary structure, quasi-native VSD-II fold, and enhanced ps-ns timescale dynamics in the micelle-solubilized domain. We modeled the structure of the VSD-II/Hm-3 complex by protein-protein docking involving binding surfaces mapped by NMR. Hm-3 binds to VSDs I and II using different modes. In VSD-II, the protruding β-hairpin of Hm-3 interacts with the S1-S2 extracellular loop, and the complex is stabilized by ionic interactions between the positively charged toxin residue K24 and the negatively charged channel residues E604 or D607. We suggest that Hm-3 binding to these charged groups inhibits voltage sensor transition to the activated state and blocks the depolarization-activated gating pore currents. Our results indicate that spider toxins represent a useful hit for periodic paralyses therapy development and may have multiple structurally different binding sites within one NaV molecule.

Keywords: Channelopathies, Sodium channel, gating modifier toxin, NMR spectroscopy, Cell-free expression, Combinatorial selective labeling, ligand-receptor interactions

Received: 25 Apr 2019; Accepted: 26 Jul 2019.

Edited by:

Frank Bernhard, Goethe University Frankfurt, Germany

Reviewed by:

Christin Schroeder, Institute for Molecular Bioscience, University of Queensland, Australia
Arun Samidurai, Virginia Commonwealth University, United States  

Copyright: © 2019 Myshkin, Mannikko, Krumkacheva, Kulbatskii, Chugunov, Berkut, Paramonov, Shulepko, Fedin, Hanna, Kullmann, Bagryanskaya, Arseniev, Kirpichnikov, Lyukmanova, Vassilevski and Shenkarev. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Zakhar O. Shenkarev, Institute of Bioorganic Chemistry (RAS), Moscow, 117997, Moscow Oblast, Russia, zakhar-shenkarev@yandex.ru