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 Yu Myshkin,Antonina A Berkut,Zakhar O Shenkarev,Alexander S Arseniev,Mikhail P Kirpichnikov,Ekaterina N Lyukmanova,Anton O Chugunov,Mikhail A Shulepko,Roope Männikkö,Dmitrii S Kulbatskii,Alexander A Vassilevski,Michael G Hanna,Elena G Bagryanskaya,Alexander S Paramonov,Olesya A Krumkacheva,Dimitri M Kullmann,Matvey V Fedin

doi:10.3389/fphar.2019.00953

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.

Highlights

Voltage-gated sodium channels (NaV) together with potassium and calcium channels form a large P-loop superfamily of integral membrane proteins (IMPs) (Hille, 2001). α-Subunits of eukaryotic NaV channels have modular architecture and are composed of four homologous repeats (Figure 1A), each closely related to a subunit of homotetrameric voltage-gated potassium channels (KV) or bacterial NaV channels
Further screening of Hm-3 on gating charge mutant channels revealed that gating pore currents through the voltage-sensing domain (VSD)-II R3 (R675G) mutant were suppressed (Figure 2)
Analogous to VSD-I R3G, the gating pore currents of VSD-II R3G are increased at depolarized voltages

Summary

Introduction

Voltage-gated sodium channels (NaV) together with potassium and calcium channels form a large P-loop superfamily of integral membrane proteins (IMPs) (Hille, 2001). α-Subunits of eukaryotic NaV channels have modular architecture and are composed of four homologous repeats (Figure 1A), each closely related to a subunit of homotetrameric voltage-gated potassium channels (KV) or bacterial NaV channels. Mutations of conserved S4 arginine residues in the skeletal muscle NaV1.4 channel (SCN4A) trigger leak currents through the VSDs, known as “gating pore currents” or “ω-currents” that are induced in addition to the main pore or α-currents (Groome et al, 2018). Mutation of the outer R1 or R2 residues leads to gating pore currents that are activated at resting potentials or hyperpolarization (Sokolov et al, 2007; Struyk and Cannon, 2007; Struyk et al, 2008), whereas replacements of the inner R3 residues induce depolarization-activated currents (Sokolov et al, 2008)

Methods

Results

Conclusion