Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking
Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking
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DOI：
10.1074/jbc.m602018200
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发表时间：
2006-09-22
影响因子：
4.8
通讯作者：
Oiki, Shigetoshi
中科院分区：
文献类型：
--
作者：
Iwamoto, Masayuki;Shimizu, Hirofumi;Oiki, Shigetoshi
KcsA是第一个发现分子结构的钾离子通道。然而，高分辨率的结构信息仅限于跨膜域，并且完整的KcsA通道的动态图像仍未解决。我们开发了一种新的方法来研究蛋白质的表面结构，并应用这种方法来研究KcsA通道的全长。在25个位点引入单半胱氨酸取代，并通过表面等离子体共振测量来评估这些突变通道对平坦金板裸露表面的特异性反应。表面等离子体共振信号显示，c端突变体的暴露程度最高。当KcsA通道门在pH 7.5下保持关闭时，暴露程度在细胞质（CP）和n端结构域的连续位点上呈现周期性模式。这表明这些延伸采用α螺旋结构。当通道在pH 4.0时被主动门控时，CP结构域的许多位点暴露出来。与pH 7.5下的刚性结构相比，这些结果表明，活性门控使CP结构域变得松散。M2螺旋的c端端是栅极的活动部分，在pH 4.0时略微暴露在外表面。通过添加通道阻滞剂四丁基铵，栅极进一步暴露。这表明在主动门控中，四丁基铵保持门打开，而不是被困在中心腔中。
KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.