Optimized expression and purification of NavAb provide the structural insight into the voltage dependence

FEBS Lett. 2018 Jan;592(2):274-283. doi: 10.1002/1873-3468.12955. Epub 2018 Jan 9.

Abstract

Voltage-gated sodium channels are crucial for electro-signalling in living systems. Analysis of the molecular mechanism requires both fine electrophysiological evaluation and high-resolution channel structures. Here, we optimized a dual expression system of NavAb, which is a well-established standard of prokaryotic voltage-gated sodium channels, for E. coli and insect cells using a single plasmid vector to analyse high-resolution protein structures and measure large ionic currents. Using this expression system, we evaluated the voltage dependence and determined the crystal structures of NavAb wild-type and two mutants, E32Q and N49K, whose voltage dependence were positively shifted and essential interactions were lost in voltage sensor domain. The structural and functional comparison elucidated the molecular mechanisms of the voltage dependence of prokaryotic voltage-gated sodium channels.

Keywords: protein expression; structural physiology; voltage-gated sodium channel.

Publication types

  • Letter
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / growth & development*
  • Gene Expression*
  • Models, Molecular
  • Mutation
  • Plasmids / genetics
  • Protein Conformation
  • Sf9 Cells
  • Voltage-Gated Sodium Channels / chemistry*
  • Voltage-Gated Sodium Channels / genetics*
  • Voltage-Gated Sodium Channels / metabolism

Substances

  • Voltage-Gated Sodium Channels

Associated data

  • PDB/5YUA
  • PDB/5YUB
  • PDB/5YUC
  • PDB/3RVY
  • PDB/5VB2
  • PDB/4EKW