The roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel
The roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel
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DOI：
10.1074/jbc.m212973200
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发表时间：
2003-04-11
影响因子：
4.8
通讯作者：
Wray, D
中科院分区：
文献类型：
--
作者：
Ju, M;Stevens, L;Wray, D
人类和大鼠形式的Kv2.1通道在跨膜区域具有相同的氨基酸，仅在N-和C-末端细胞内区域不同。大鼠Kv2.1比人类Kv2.1激活更快。在这里，我们已经研究了N-和C-末端残基的作用，确定这两个通道之间的激活动力学的差异。为此，我们构建了两个通道之间的突变体和嵌合体，在卵母细胞中表达它们，并通过双电极电压钳位记录电流。在N-末端区域中，大鼠通道中的突变Q67 E相对于大鼠野生型显示出活化减慢，而人通道中的突变D 75 E显示出比人野生型更快的活化。在C-末端区域中，我们发现氨基酸740-853（“CTA”结构域）区域内的一些残基也参与决定活化动力学。电生理数据还表明N和C末端之间的相互作用。通过使用谷胱甘肽S-转移酶（GST）融合蛋白与Kv2.1的N末端，我们证明结合到Kv2.1的C末端，直接证实了这种相互作用。总之，这些数据表明，暴露的残基在T1结构域的N端，以及CTA结构域的C端，是重要的，在确定通道激活动力学，这些N-和C-末端区域相互作用。
The human and rat forms of the Kv2.1 channel have identical amino acids over the membrane-spanning regions and differ only in the N- and C-terminal intracellular regions. Rat Kv2.1 activates much faster than human Kv2.1. Here we have studied the role of the N- and C-terminal residues that determine this difference in activation kinetics between the two channels. For this, we constructed mutants and chimeras between the two channels, expressed them in oocytes, and recorded currents by two-electrode voltage clamping. In the N-terminal region, mutation Q67E in the rat channel displayed a slowing of activation relative to rat wild type, whereas mutation D75E in the human channel showed faster activation than human wild type. In the C-terminal region, we found that some residues within the region of amino acids 740-853 ("CTA" domain) were also involved in determining activation kinetics. The electrophysiological data also suggested interactions between the N and C termini. Such an interaction was confirmed directly by using a glutathione S-transferase (GST) fusion protein with the N terminus of Kv2.1, which we showed to bind to the C terminus of Kv2.1. Taken together, these data suggest that exposed residues in the T1 domain of the N terminus, as well as the CTA domain in the C terminus, are important in determining channel activation kinetics and that these N- and C-terminal regions interact.