A Simple Assay to Evaluate the Function of Human Connexin Hemichannels Expressed in Escherichia coli that Can Be Used for Drug Discovery and Mutant Analysis.
A Simple Assay to Evaluate the Function of Human Connexin Hemichannels Expressed in Escherichia coli that Can Be Used for Drug Discovery and Mutant Analysis.
复制标题
评估大肠杆菌中表达的人连接蛋白半通道功能的简单测定，可用于药物发现和突变分析。
DOI：
10.1002/cpph.68
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发表时间：
2019
影响因子：
--
通讯作者：
Altenberg,GuillermoA
中科院分区：
文献类型：
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作者：
Fiori,MarianaC;Cuello,LuisG;Altenberg,GuillermoA
在包括耳聋、中风和心肌梗死在内的许多疾病中，连接蛋白半通道活性的异常增加都会导致细胞损伤，因此半通道是潜在的重要治疗靶点。不幸的是，现有的半通道抑制剂并不是特异的，而且大多数都是有毒的。由于缺乏一种简单且成本效益高的筛选方法，半通道抑制剂的发现变得困难。在这里，我们提出了一种优化的检测方法，其中人连接蛋白在缺乏钾吸收的基因修饰的大肠杆菌细胞(LB2003细胞)中表达。这些细胞不能在低钾介质中生长，半通道功能是通过逆转无生长表型来检测的。由于功能半通道对钾具有渗透性，它们允许钾的吸收和细胞生长。细菌在低钾介质中生长的简单读数区分了功能性半管(生长)和抑制半管(不生长)。这种检测方法简单、可靠、价廉，并且很容易扩展到高通量的多井平台。©2019 John Wiley&Sons，Inc.基本方案1：制备抗卡那霉素的合格LB2003细胞基本方案2：生长互补分析支持方案：评估潜在连接蛋白半通道抑制剂的细胞毒性效应
Abnormally increased activity of connexin hemichannels contributes to cell damage in many disorders, including deafness, stroke, and cardiac infarct, and therefore hemichannels constitute a potentially important therapeutic target. Unfortunately, the available hemichannel inhibitors are not specific and most are toxic. The absence of a simple and cost‐effective screening assay has made the discovery of hemichannel inhibitors difficult. Here, we present an optimized assay where human connexins are expressed in genetically modifiedEscherichia colicells deficient in potassium uptake (LB2003 cells). These cells cannot grow in low‐potassium medium, and hemichannel function is assayed by the reversion of the no‐growth phenotype. Since functional hemichannels are permeable to potassium, they allow for its uptake and cell growth. The simple reading of bacterial growth in low‐potassium medium distinguishes functional hemichannels (growth) from those inhibited (no growth). This assay is simple, robust, inexpensive, and reliable, and is easily scaled to high‐throughput multiwell platforms. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: Preparation of competent LB2003 cells resistant to kanamycinBasic Protocol 2: Growth complementation assaySupport Protocol: Evaluation of cytotoxic effects of potential connexin hemichannel inhibitors