Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology PAS, Pasteur str. 3, Warsaw 02-093, Poland.
Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology PAS, Pasteur str. 3, Warsaw 02-093, Poland.
Biochim Biophys Acta Biomembr. 2021 Apr 1;1863(4):183555. doi: 10.1016/j.bbamem.2021.183555. Epub 2021 Jan 11.
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 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.
表达、纯化和功能重建哺乳动物离子通道通常具有挑战性。由于与蛋白质环境无关，并且缺乏共纯化内源性蛋白质（例如，影响通道活性的辅助通道亚基）的风险，因此在细菌中异源表达哺乳动物通道可能具有优势。此外，由于它们的细胞内定位，例如线粒体通道，直接记录通道活性可能具有挑战性。纯化通道的活性可以通过电生理技术（如平面脂质双层 (PLB)）在单分子水平上进行表征。在这项工作中，我们描述了一种简单的方法来完成在大肠杆菌中表达的单个肾髓质外钾通道 ROMK 的 PLB 记录。我们专注于 ROMK2 同工型，它在线粒体中含量低，可能负责 mitoK 活性。我们筛选了最佳构建体，以表达带有 6xHis 标签的密码子优化的 ROMK 蛋白，用于蛋白质纯化。该策略涉及使用最佳苯乙烯-马来酸 (SMA) 共聚物，其形成所谓的聚合物纳米盘，以溶解和纯化含有 ROMK 的 SMA 脂质颗粒 (SMALPs)，这些颗粒可与 PLB 融合。重建的 ROMK 通道表现出离子选择性、整流和药理学特性，与之前关于 ROMK 通道的工作一致。
