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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

Myshkin, MY; Mannikko, R; Krumkacheva, OA; Kulbatskii, DS; Chugunov, AO; Berkut, AA; Paramonov, AS; ... Shenkarev, ZO; + view all (2019) 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. Frontiers in Pharmacology , 10 , Article 953. 10.3389/fphar.2019.00953. Green open access

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Abstract

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 nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy study of isolated VSD-II in zwitterionic dodecylphosphocholine/lauryldimethylamine-N-oxide or dodecylphosphocholine 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 electron paramagnetic resonance 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 ß-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.

Type: Article
Title: 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
Open access status: An open access version is available from UCL Discovery
DOI: 10.3389/fphar.2019.00953
Publisher version: https://doi.org/10.3389/fphar.2019.00953
Language: English
Additional information: Copyright © 2019 Myshkin, Männikkö, 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) (http://creativecommons.org/licenses/by/4.0/). 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.
Keywords: channelopathies, sodium channel, gating modifier, NMR spectroscopy, cell-free expression, combinatorial selective labeling, ligand-receptor interactions
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Clinical and Experimental Epilepsy
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Department of Neuromuscular Diseases
URI: https://discovery.ucl.ac.uk/id/eprint/10082271
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