PIP2诱导打开内向整流钾通道门控的分子机理的研究

Inwardly rectifying K+ (Kir) channels comprise one family of K+ selective ion channels. One prominent feature of Kir channels is their regulation by the signaling lipid phosphatidylinositol bisphosphate (PIP2). Alterations in channel-PIP2 interactions change Kir single-channel gating behavior. Channelopathies, like Bartter’s syndromes are associated with mutations on Kir channels, which affect channel-PIP2 interactions. With the crystal structures of Kir-PIP2 complexes, we have known the binding site of PIP2 on Kir channels and the interactions formed between PIP2 and Kir channels. However, it remains unclear how PIP2 binding induces the channel structural transition from the closed to the open state. We plan to use molecular dynamics simulations based on the Kir2.2 and Kir3.1 chimera to study the molecular events that occur from PIP2 binding to channel gating. The simulation systems will be constructed based on the open and closed state of the crystal structures in the absence and presence of PIP2. We will focus on analyzing of residue interaction network in each system and thoroughly comparing changes of networks among systems. The explicit changes in networks of interactions that stabilize either the closed (in the absence of PIP2) or open (in the presence of PIP2) state will emerge and molecular mechanism of how PIP2 induce the opening of the gate can be therefore proposed. Our preliminary study on Kir3.1 chimera based on this methods have already detailed the molecular mechanism of how PIP2 induce the opening of the G-loop gate. We then will concentrate on other gates, helix bundle crossing gate and selectivity filter of the Kir2.2 and Kir3.1 chimera. Kir channels are potentially important targets for pharmaceutical agents as their diverse physiological functions. Because the cardiac Kir (Kir2 subfamily) require PIP2 for normal activity, obtaining a structural understanding of how PIP2 regulates their activity is potentially a powerful step toward designing drugs for cardiac arrhythmias.

内向整流钾通道（Kir）依赖磷脂酰肌醇二磷酸（PIP2）维持其正常活性。PIP2和Kir的相互作用与通道门控机制密切相关，并涉及巴特综合症等疾病。Kir-PIP2复合物晶体结构已知，然而，PIP2与通道结合后怎样诱导通道开放的分子机制仍不明确。本项目拟通过分子动力学模拟的方法研究Kir2.2和Kir3.1嵌合体在PIP2诱导下从关闭到打开的分子机制。我们将分别从门控的关闭态和开放态结构出发，构建有PIP2和没有PIP2的对照体系，着重分析在动力学过程中残基相互作用网络的变化情况，由此提出PIP2诱导通道构象变化的机理，找出稳定关闭/开放态的门控的关键残基相互作用。前期利用这种方法对Kir3.1嵌合体G-loop门控的研究给出了门控打开的详细分子机制。我们将继续探索这两个通道其他门控的作用机制。Kir2是潜在药物作用靶点。搞清楚PIP2对Kir2的门控机制对研发抗心律不齐药物具有指导意义。

内向整流型钾离子通道Kir依赖磷脂酰肌醇PIP2维持其正常活性。Kir与PIP2之间的相互作用涉及巴特尔综合征等离子通道疾病。近年来Kir离子通道晶体结构的获得，十分有利于利用分子动力学模拟研究PIP2诱导Kir通道打开的分子机理。本项目着重探索PIP2诱导打开Kir G-loop门控和HBC门控的分子机制。我们从闭合G-loop+PIP2出发，诱导G-loop至半开放态，对比分析其与闭合态和开放态，发现开放态时，G-loop呈正方形，四个亚基界面为unlatched态（一个亚基的βM与相邻亚基的N端成β片层）；闭合态时，G-loop呈长条状，四个亚基界面为latched态（N端成loop，不与βM作用）。半开放态时，G-loop打开，亚基界面中有三个成unlatched态一个成latched态。通过残基相互作用网络的变化和与PIP2成盐桥情况，我们提出了PIP2诱导G-loop打开的分子机理：在没有 PIP2 作用时，通道的 N端稳定非活性状态的CD-loop，CD-loop与G-loop的联系较为松散，闭合的G-loop构象由保守盐桥E304-R313所稳定；PIP2出现后首先定位 N端，减弱N端与CD-loop的作用，同时使G-loop直接与N端发生联系；之后CD-loop发生构象变化，远离N端，加强与G-loop的联系，破坏G-loop与N端的作用，G-loop 由闭合转向打开，此时N端失去了与CD-loop和G-loop的作用，转而与βM形成β片层从而被稳定。CD-loop上的正电残基被 PIP2 吸引，从而进一步稳定了活性状态下的CD-loop处于打开状态的G-loop构象。我们引入了突变M170P帮助HBC门控打开。HBC的打开是由跨膜螺旋TM1和TM2的逆时针旋转以及TM2在G169位置弯折的共同作用。HBC的打开需要PIP2的存在以及G-loop的先行开放，两者缺一不可。HBC开放态时孔径直径6Å，闭合态3Å。估算的HBC门控开放的自由能差为1.4kcal/mol. HBC和G-loop同时开放的情况下，我们观测到了钾离子由胞外流入胞内的过程。我们找出了影响门控打开的关键因素，获得开放态的Kir通道模型，以及从闭合到开放的动力学过程，有助于理解一些离子通道疾病的治病突变的分子机制。此外，也尝试探索了一套基于分子动力学模拟的分析蛋白质别构效应的方法。