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Chem Phys Lipids. 2019 Jan 31;219:50-57. doi: 10.1016/j.chemphyslip.2019.01.013. [Epub ahead of print]

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

Author information

1
Department of Biology, Moscow Lomonosov State University, 119234, Moscow, Russia.
2
Department of Physics, University of Osnabrück, 49069, Osnabrück, Germany.
3
Department of Biology, Moscow Lomonosov State University, 119234, Moscow, Russia; Laboratory of Cardiac Physiology, Institute of Physiology, Komi Science Center, Ural Branch, Russian Academy of Sciences, Syktyvkar, Russia.
4
INSERM, CNRS, l'Institut du Thorax, Université de Nantes, 44007, Nantes, France.
5
Department of Biology, Moscow Lomonosov State University, 119234, Moscow, Russia. Electronic address: sokolova@mail.bio.msu.ru.

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

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