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Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels.

机译:微生物内向整流钾离子通道超家族的克隆和功能鉴定。

摘要

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.
机译:最近,我们通过两个同源原核直系同源物(KirBac1.1和KirBac3.1)的X射线晶体结构提高了我们对K +通道哺乳动物内向整流家族的了解。但是,这些KirBac通道的功能特性仍然知之甚少。为了解决这个问题,我们从多种不同的原核生物和简单的单细胞真核生物中克隆并鉴定了编码KirBac直向同源物的基因。然后通过在K +摄取缺陷型大肠杆菌(TK2420)中的生长互补来检查这些KirBac的功能特性。某些KirBac基因表现出强劲的生长互补性,而另一些则不互补或表现出温度依赖性互补,包括KirBac1.1和KirBac3.1。在某些情况下,KirBac表达对大肠杆菌的生长也有毒。 KirBac家族对K +通道阻滞剂Ba2 +和Cs +表现出一定的敏感性,以及它们在极低K +的培养基上生长的能力方面的差异,因此证明了其渗透性能的重大差异。这些结果揭示了微生物KirBac基因功能上多样化的超家族的存在,并为此类K +通道的结构和功能分析提供了极好的资源。此外,本研究中使用的互补测定法为一系列传统方法难以研究的原核K +通道的功能表征提供了一种简单而强大的方法。

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