Yuchi Zhiguang, Pau Victor P T, Yang Daniel S C
Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Canada.
FEBS J. 2008 Dec;275(24):6228-36. doi: 10.1111/j.1742-4658.2008.06747.x. Epub 2008 Nov 7.
The prokaryotic potassium channel from Streptomyces lividans, KcsA, is the first channel that has a known crystal structure of the transmembrane domain. The crystal structure of its soluble C-terminal domain, however, still remains elusive. Biophysical and electrophysiological studies have previously implicated the essential roles of the C-terminal domain in pH sensing and in vivo channel assembly. We examined this functional assignment by replacing the C-terminal domain with an artificial tetramerization domain, GCN4-LI. The expression of KcsA is completely abolished when its C-terminal domain is deleted, but it can be rescued by fusion with GCN4-LI. The secondary and quaternary structures of the hybrid channel are very similar to those of the wild-type channel according to CD and gel-filtration analyses. The thermostability of the hybrid channel at pH 8 is similar to that of the wild-type but is insensitive to pH changes. This supports the notion that the pH sensor of KcsA is located in the C-terminal domain. The result obtained in the present study is in agreement with the proposed functions of the C-terminal domain and we show that the channel assembly role of the C-terminal domain can be substituted with a non-native tetrameric motif. Because tetramerization domains are found in different families of potassium channels and their presence often enhances the expression of channels, replacement of the elusive C-terminal domains with a known tetrameric scaffold could potentially assist the expression of other potassium channels.
来自淡紫链霉菌的原核钾通道KcsA，是首个其跨膜结构域具有已知晶体结构的通道。然而，其可溶性C端结构域的晶体结构仍然未知。此前的生物物理和电生理研究表明，C端结构域在pH传感和体内通道组装中起着重要作用。我们通过用人工四聚化结构域GCN4-LI替换C端结构域来检验这一功能归属。当KcsA的C端结构域被删除时，其表达完全丧失，但通过与GCN4-LI融合可以挽救。根据圆二色光谱（CD）和凝胶过滤分析，杂合通道的二级和四级结构与野生型通道非常相似。杂合通道在pH 8时的热稳定性与野生型相似，但对pH变化不敏感。这支持了KcsA的pH传感器位于C端结构域的观点。本研究获得的结果与C端结构域的推测功能一致，并且我们表明C端结构域的通道组装作用可以被非天然的四聚基序替代。由于四聚化结构域存在于不同家族的钾通道中，并且它们的存在通常会增强通道的表达，用已知的四聚支架替换难以捉摸的C端结构域可能有助于其他钾通道的表达。
