Dynamic interaction of S5 and S6 during voltage-controlled gating in a potassium channel.
Dynamic interaction of S5 and S6 during voltage-controlled gating in a potassium channel.
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DOI：
10.1085/jgp.118.2.157
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发表时间：
2001-08
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秀丽隐杆线虫exp-2 K+通道基因中的功能获得性突变由通道的第六跨膜区段中的半胱氨酸至酪氨酸的变化（C480 Y）引起（Davis，M.W.，R. Fleischhauer，J.A. Dent，R.H. Joho和L.埃弗里1999.科学286：2501-2504）。与野生型EXP-2通道相反，纯四聚体C480 Y突变体通道甚至在-160 mV时也是开放的，这解释了纯合突变体的致死性。我们在K+通道KcsA的3-D支架上模拟了EXP-2的结构。在C480 Y突变体中，酪氨酸480从S6突出到S5附近，表明大体积侧链可以为S6的旋转提供空间位阻，已经提出S6的旋转伴随开-闭状态转变（Perozo，E.，D.M.科尔特斯和L.G.奎罗1999.科学285：73-78）。我们测试了这样的假设，即只有在位置480处的小侧链允许通道关闭，但是大的侧链将通道捕获在打开状态。具有小侧链取代（Gly和Ser）的突变体表现得像野生型;相反，大侧链取代（Trp、Phe、Leu、Ile、瓦尔和His）产生的通道在负电位为−120 mV时传导K+离子。孔模型中S6中位置480处的侧链接近S5中的保守甘氨酸（G421）并可与其相互作用。用庞大的侧链取代G421也导致被捕获在活性状态的通道，这表明S5和S6在电压依赖性开放-关闭状态转变期间彼此相互作用，并且庞大的侧链阻止永久通道关闭所需的动态变化。单通道记录显示，突变体通道在负膜电位下频繁打开，表明它们未能达到持久的，即，稳定的封闭状态我们的数据支持“双门模型”，其中孔门负责短暂的电压无关的开口，而单独定位的电压激活的门（Liu，Y.，和R.H.约翰1998. Pflügers Arch. 435：654-661）。
A gain-of-function mutation in the Caenorhabditis elegans exp-2 K+-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W., R. Fleischhauer, J.A. Dent, R.H. Joho, and L. Avery. 1999. Science. 286:2501–2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at −160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K+ channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73–78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K+ ions at potentials as negative as −120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact with a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a “two-gate model” with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H. Joho. 1998. Pflügers Arch. 435:654–661).