Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels
Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels
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DOI：
10.1152/physiolgenomics.00026.2006
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发表时间：
2006-06-16
影响因子：
4.6
通讯作者：
Tucker, Stephen J.
中科院分区：
文献类型：
--
作者：
Sun, Si;Gan, Jo Han;Tucker, Stephen J.
最近，我们通过两个同源原核直系同源物（KirBac1.1和KirBac3.1）的X射线晶体结构，对哺乳动物内向整流钾离子通道家族（Kir家族）的理解有了进展。然而，这些KirBac通道的功能特性仍然知之甚少。为了解决这个问题，我们克隆和表征编码KirBac同源基因从各种不同的原核生物和一个简单的单细胞真核生物。这些KirBacs的功能特性，然后检查在大肠杆菌（TK2420）的K+摄取缺陷菌株的生长互补。而一些KirBac基因表现出强大的生长互补，其他要么不互补或显示温度依赖性互补，包括KirBac1.1和KirBac3.1。在某些情况下，KirBac表达对E.杆菌KirBac家族对K+通道阻断剂Ba 2+和Cs+表现出一系列的敏感性，以及它们在非常低的K+介质上生长的能力的差异，从而证明了它们的渗透特性的主要差异。这些结果揭示了微生物KirBac基因的功能多样的超家族的存在，并提出了这类K+通道的结构和功能分析的一个很好的资源。此外，在这项研究中使用的互补试验提供了一个简单而强大的方法，一系列的原核K+通道的功能表征，难以通过传统的方法进行研究。
Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.