【作者】 郭蕊；

【导师】 叶升；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2016， 博士

【摘要】 钾离子通道能够控制钾离子选择性地并且高速地流入和流出细胞来改变细胞内外电压,从而使得细胞对外界刺激产生瞬间反应。钾离子通道的离子选择筛中的四个钾离子结合位点只能同时容纳两个钾离子,钾离子在离子选择筛中以1,3和2,4两种相互平衡的状态存在。钡离子是唯一一个能够阻断钾离子通道的碱土金属元素,钡离子和钾离子大小类似(钡的离子半径为1.35A,钾为1.33 A),然而钡离子却携带双倍的电荷。钡离子的大小使得它很容易进入钾离子通道的离子选择筛,然而它所携带的电荷使它在离子选择筛中的结合过于紧密,因而导致钡离子对钾离子通道的阻断效应。离子通道研究领域的先驱,布兰迪斯大学(Brandeis University)的Christopher Miller博士在上世纪八十年代通过对钾通道的钡阻断进行电生理学研究发现钾离子对于钡离子从钾通道逃逸存在三个效应：外部锁定效应(external lock-in effect)、增强效应(enhancement effect)和内部锁定效应(internal lock-in effect),他还前瞻性地推理出钾离子通道的结构,并在十几年后被钾离子通道的晶体结构所验证。然而钡离子和钾离子在钾离子通道中是如何相互作用而导致这些效应,至今仍然是离子通道研究领域争议的热点。我们以MthK钾离子通道为模型,通过多学科交叉,结合X-射线晶体学和电生理学两种研究手段,探讨了钡离子和钾离子在MthK钾离子通道内的相互作用,对上述三个效应提出了一个相应的模型。在没有钡离子时,钾离子在钾离子通道的离子选择筛中以1,3-和2,4-两种相互平衡的状态存在,此时,钾离子通道能够自由通透钾离子。钡离子从胞内侧进入钾离子通道离子选择筛,结合在4、3或2位点阻断钾离子通道。钡离子可以向外或向内逃逸出使钾离子通道畅通。钾离子从外侧进入位点1,将钡离子推至位点4,阻止钡离子向外逃逸(外部锁定效应)；外侧钾离子浓度高时能将钾离子推至位点2,从而增强钡离子向内逃逸(增强效应)；内侧的钾离子在中央空穴能阻止钡离子向内逃逸(内部锁定效应)。更多还原

【Abstract】 Potassium channels control the electric potential across cell membranes. They catalyze the rapid, selective diffusion of K+ions down their electrochemical gradient. Under physiological conditions, the K+selectivity filter usually contains two resident K+ ions separated by a water molecule. The ion pair moves back and forth in a concerted manner between the 1,3 and 2,4 configuration.Ba2+is the only alkaline earth metal that blocks K+channels, presumably because it uniquely shares high similarity with the alkali metal K+in terms of the ionic radius (1.35 A of Ba2+vs.1.33 A of K+) while doubling the charge. The similar size allows Ba2+ions to fit into the K+channel selectivity filter. However, the charge apparently causes tight binding, preventing the rapid flow of K+. Doctor Christopher Miller from Brandeis University, pioneer in the ion channel field found Ba2+have 3 effects on the K+channel through electrophysiology studies, that is external lock-in effect, enhancement effect and internal lock-in effect. He also supposed the structure of K+channel which was solved by x-ray crystallography. However, how Ba2+and K+interactions lead to these effects remains to be unclear, so further studies are needed.In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba2+with permeant ions within the ion conduction pathway of the MthK K+channel and proposed a model. Without Ba2+, two K+ions bind equivalently in the 1,3 and 2,4 configurations. With the addition of Ba2+from the internal side, Ba2+ enters the filter and initiates block by occupying the S4 site. The electrostatic repulsion between Ba2+and K+allows the Ba2+ion to effectively drive out K+ions from the filter, leaving a single Ba2+ion alternating between S2, S3, and S4 sites and blocking the passage of K+ions. Ba2+ions may leave the filter either by dissociation back to the internal side or proceeding to the external side. The addition of external K+in low concentration causes K+ion binding at its high affinity site 1, locking Ba2+at site 4 and preventing Ba2+from exiting to the external side (external lock-in effect). Although external K+in high concentration allows the K+ion to enter site 2, it forces Ba2+ion dissociation to the internal side by the electrostatic repulsion (enhancement effect). The internal K+, or other permeant ions, can occupy the central cavity, locking the Ba2+ion mainly at site 2 and preventing the Ba2+ion from exiting to the internal side (internal lock-in effect).更多还原