Detergent-free solubilization of human Kv channels expressed in mammalian cells
Autor(en): | Karlova, M. G. Voskoboynikova, N. Gluhov, G. S. Abramochkin, D. Malak, O. A. Mulkidzhanyan, A. Loussouarn, G. Steinhoff, H. -J. Shaitan, K. V. Sokolova, O. S. |
Stichwörter: | Affinity purification; Biochemistry & Molecular Biology; Biophysics; CRYO-EM STRUCTURE; DEPENDENT K+ CHANNEL; Dynamic light scattering; Electron microscopy; ELECTRON-MICROSCOPY; GATED POTASSIUM CHANNELS; GENE; hKCNE1-hKCNQ1 complex; Human Kv channels; ION-CHANNEL; MALEIC ACID COPOLYMER; MEMBRANE-PROTEINS; NANODISCS; RECONSTITUTION; SMALP | Erscheinungsdatum: | 2019 | Herausgeber: | ELSEVIER IRELAND LTD | Journal: | CHEMISTRY AND PHYSICS OF LIPIDS | Volumen: | 219 | Startseite: | 50 | Seitenende: | 57 | Zusammenfassung: | 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 alpha-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an a-subunit hKCNQ1 and its regulatory transmembrane KCNE1 beta-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. |
ISSN: | 00093084 | DOI: | 10.1016/j.chemphyslip.2019.01.013 |
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geprüft am 16.05.2024