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Solubilization, purification, and functional reconstitution of human ROMK potassium channel in copolymer styrene-maleic acid (SMA) nanodiscs.

Authors
  • Krajewska, Milena1
  • Koprowski, Piotr2
  • 1 Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology PAS, Pasteur str. 3, Warsaw 02-093, Poland. , (Poland)
  • 2 Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology PAS, Pasteur str. 3, Warsaw 02-093, Poland. Electronic address: [email protected] , (Poland)
Type
Published Article
Journal
Biochimica et biophysica acta. Biomembranes
Publication Date
Jan 11, 2021
Volume
1863
Issue
4
Pages
183555–183555
Identifiers
DOI: 10.1016/j.bbamem.2021.183555
PMID: 33444624
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Expression, purification, and functional reconstitution of mammalian ion channels are often challenging. Heterologous expression of mammalian channels in bacteria can be advantageous due to unrelated protein environment and the lack of risk of copurification of endogenous proteins, e.g., accessory channel subunits that can influence the channel activity. Also, direct recording of channel activity could be challenging due to their intracellular localization like in the case of mitochondrial channels. The activity of purified channels can be characterized at the single-molecule level by electrophysiological techniques, such as planar lipid bilayers (PLB). In this work, we describe a simple approach to accomplish PLB recording of the activity of single renal outer medullary potassium channels ROMK expressed in E. coli. We focused on the ROMK2 isoform that is present at low levels in the mitochondria and can be responsible for mitoKATP activity. We screened for the best construct to express the codon-optimized ROMK proteins with a 6xHis tag for protein purification. The strategy involved the use of optimal styrene-maleic acid (SMA) copolymer, which forms so-called polymer nanodiscs, to solubilize and purify ROMK-containing SMA lipid particles (SMALPs), which were amenable for fusion with PLB. Reconstituted ROMK channels exhibited ion selectivity, rectification, and pharmacological properties, which are in agreement with previous work on ROMK channels. Copyright © 2021. Published by Elsevier B.V.

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