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

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 mitoK AT p 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.

Automated summary

The expression, purification, and functional reconstitution of mammalian ion channels can be challenging, especially when dealing with intracellularly localized channels like mitochondrial ones. In this study, a simple approach was described to record the activity of single renal outer medullary potassium channels ROMK expressed in Escherichia coli using planar lipid bilayers. By optimizing construct and utilizing styrene-maleic acid copolymer, ROMK proteins were successfully purified and reconstituted for further characterization, exhibiting ion selectivity, rectification, and pharmacological properties consistent with previous research on ROMK channels.