An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels
An Escherichia coli-Based Assay to Assess the Function of Recombinant Human Hemichannels
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DOI：
10.1177/1087057116675321
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发表时间：
2017-02-01
期刊：
影响因子：
3.1
通讯作者：
Cuello, Luis G.
中科院分区：
文献类型：
--
作者：
Krishnan, Srinivasan;Fiori, Mariana C.;Cuello, Luis G.
连接蛋白形成介导细胞间通讯的差距连接通道，并且还形成存在于质膜处的半通道。半通道可渗透小的亲水性化合物，包括参与自分泌和旁分泌信号传导的分子。异常的半通道开放导致或促成常见人类病症中的细胞损伤（例如，心脏梗塞、脑血管意外、耳聋、皮肤病和白内障），因此是潜在的药理学靶标。有用的半通道抑制剂的发现部分地受到缺乏合适的高通量功能测定的阻碍。在这里，我们开发并表征了一种检测方法，该方法可用于评估由在遗传修饰的大肠杆菌菌株中表达的人连接蛋白形成的半通道的功能。LB 2003细胞缺乏三种关键的K+摄取转运机制，不能在低[K+]培养基中生长，但Cx26、Cx43或Cx46的表达挽救了它们的生长缺陷（生长互补）。我们开发了一种简单、廉价、易于扩展、可重复和灵敏的检测方法，该方法可用于基于小化合物文库分析发现新的更好的半通道抑制剂。
Connexins form the gap junctional channels that mediate cell-to-cell communication, and also form hemichannels present at the plasma membrane. Hemichannels are permeable to small hydrophilic compounds, including molecules involved in autocrine and paracrine signaling. An abnormal hemichannel opening causes or contributes to cell damage in common human disorders (e.g., cardiac infarct, cerebrovascular accidents, deafness, skin diseases, and cataracts) and is therefore a potential pharmacological target. The discovery of useful hemichannels inhibitors has been hampered in part by the lack of suitable high-throughput functional assays. Here, we developed and characterized an assay useful to assess the function of hemichannels formed by human connexins expressed in a genetically modified Escherichia coli strain. The LB2003 cells, devoid of three key K+ uptake transport mechanisms, cannot grow in low-[K+] medium, but expression of Cx26, Cx43, or Cx46 rescues their growth defect (growth complementation). We developed a protocol for a simple, inexpensive, easily scalable, reproducible, and sensitive assay that should be useful for the discovery of new and better hemichannel inhibitors based on the analysis of small-compound libraries.