Detergent-free solubilization of human Kv channels expressed in mammalian cells

Chem Phys Lipids. 2019 Mar;219:50-57. doi: 10.1016/j.chemphyslip.2019.01.013. Epub 2019 Jan 31.

Abstract

Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.

Keywords: Affinity purification; Dynamic light scattering; Electron microscopy; Human Kv channels; SMALP; hKCNE1-hKCNQ1 complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • COS Cells
  • Chlorocebus aethiops
  • Cricetinae
  • Cricetulus
  • Detergents / chemistry
  • Humans
  • Microscopy, Electron
  • Patch-Clamp Techniques
  • Polystyrenes / chemistry
  • Potassium Channels, Voltage-Gated / chemistry*
  • Potassium Channels, Voltage-Gated / genetics
  • Potassium Channels, Voltage-Gated / metabolism
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Solubility

Substances

  • Detergents
  • Polystyrenes
  • Potassium Channels, Voltage-Gated
  • Protein Subunits
  • Recombinant Proteins
  • styrofoam