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_ATP 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.

哺乳动物离子通道的表达，纯化和功能重建通常具有挑战性。哺乳动物通道在细菌中的异源表达可能是有利的，这是由于无关的蛋白质环境和缺乏共纯化内源蛋白质（例如可能影响通道活性的辅助通道亚基）的风险。同样，由于其在细胞内的定位（如线粒体通道的情况），直接记录通道活性可能具有挑战性。可以通过电生理技术，例如平面脂质双层（PLB）在单分子水平上表征纯化通道的活性。在这项工作中，我们描述了一种简单的方法来完成PLB记录大肠杆菌中表达的单个肾外髓质钾通道ROMK的活性。我们专注于线粒体中低水平存在的ROMK2同工型，它可以负责mitoK _ATP活性。我们筛选了最佳的构建体，以表达具有6xHis标签的密码子优化的ROMK蛋白，以进行蛋白纯化。该策略涉及使用最佳苯乙烯-马来酸（SMA）共聚物（形成所谓的聚合物纳米圆盘）来溶解和纯化可与PLB融合的含ROMK的SMA脂质颗粒（SMALP）。重建的ROMK通道具有离子选择性，整流和药理特性，这与以前在ROMK通道上的工作一致。