Exploitation of a Pharmacological 'Master Switch' to Probe the K+ Channel Selectivity Filter Gating Mechanism

几乎每个活着的细胞都是电活性的，因为它的膜上存在电压差。然而，与我们在国内非常熟悉的电子电流不同，这种“生物电”是由带电离子(如Na+和K+)通过细胞膜上被称为“离子通道”的小孔进出细胞而产生的。这些离子运动产生的电流几乎控制或影响人体的每一个过程，从我们的运动和呼吸方式，到我们的心脏和肾脏的工作方式，到我们的思维方式，以及我们感受疼痛的方式。甚至细菌也利用其膜电位的类似变化来控制重要的过程。因此，毫不夸张地说，离子通道对地球上所有形式的生命都是必不可少的。因此，许多药物和毒物通过调节这些微小分子机器的活动来发挥作用，许多人和动物的疾病都是由于离子通道功能受损而引起的。钾(K+)选择性通道是离子通道中最大的一组；人类有80种不同的K+通道基因，K+通道几乎存在于每个活着的有机体(甚至一些病毒)中。因此，它们的结构在这些不同的生命形式中是非常保守的。为了有效地运行，所有的K+通道都需要两个关键属性：一个‘过滤器’，这样它们可以选择K+而不是其他离子，以及一个‘门’，它打开和关闭来调节离子的流动。这种开启和关闭或“门控”由许多不同的细胞信号(包括许多潜在的药物)调节。这使得K+通道成为治疗疾病的有吸引力的靶点。K+通道的第一个3D结构是在20年前确定的，揭示了K+选择性过滤器和门是如何由通道两端的两个截然不同的特征形成的。然而，最近的研究表明，在某些类型的K+通道中，正常(下)门保持打开，而所有离子流的调节都是通过同时充当过滤器和门的选择性过滤器来完成的。不幸的是，过滤器门的机制一直很难研究，因为几乎没有工具来操纵这一过程。一些计算方法可以直接研究单个离子的渗透，但这些测量通常仅限于几百万分之一秒，即使是最灵敏的电子记录也无法直接测量这个时间尺度上的离子通道活动。因此，很难将离子的运动与通道的3D结构联系起来，因此需要新的方法来解决这个问题。幸运的是，我们现在已经确定了一类新的药物，它们与过滤器门相互作用，直接激活K+的流动。有趣的是，这些药物作用于一系列不同的K+通道，因此提供了一套新的工具来操纵过滤门并剖析其作用机制。在这个项目中，我们提议开发这些令人兴奋的新工具来操纵离子通过单个离子通道的渗透。其他方法也将使我们能够将我们的测量结果与渗透的结构和计算模型进行比较。此外，这些研究可能使我们能够分类(和预测)其他K+通道药物如何与通道相互作用，以及它们可能在哪里结合。这将对针对这些渠道的新药的合理设计具有重要影响。总体而言，这一建议得到了大量数据的支持，这些数据证明了我们方法的可行性。因此，它提供了一个独特而及时的机会来理解这一最基本的生物信号传递过程，并将提高我们设计针对K+通道的新药的能力。

Almost every living cell is electrically active because a voltage difference exists across its membrane. However, unlike the electronic currents we are so familiar with domestically , this 'biological electricity' results from the movement of charged ions (like Na+ and K+) into and out of cells through tiny pores in their membrane known as 'ion channels'.These ion movements generate electrical currents that control or influence almost every process in the human body, from the way we move and breathe, the way our heart and kidneys work, to the way we think, and how we sense pain. Even bacteria use similar changes in their membrane potential to control important processes. So it is no overstatement to say that ion channels are essential for all forms of life on earth. As a consequence, a wide range of medicinal drugs and many poisons exert their effects by regulating the activity of these tiny molecular machines, and many human and animal diseases result from impaired ion channel function.Potassium (K+) selective channels are one of the largest groups of ion channels; there are >80 different human K+ channel genes and K+ channels are found in almost every living organism (and even some viruses). As a result, their structural architecture is very highly conserved across these different life forms.To operate effectively, all K+ channels need two key properties: a 'filter' so they can be selective for K+ over other ions, and a 'gate' that opens and closes to regulate the flow of ions. This opening and closing or 'gating' is regulated by many different cellular signals (including many potential drugs). This makes K+ channels attractive therapeutic targets for the treatment of disease.The first 3D structure of a K+ channel was determined exactly 20 years ago and revealed how the K+ selective filter and gate are formed by two distinct features at either end of the channel. However, more recent studies now suggest that in some types of K+ channel the normal (lower) gate remains open, and instead all regulation of ionic flow is done via the selectivity filter which acts as both filter AND gate.Unfortunately, the mechanism of filter gating has been difficult to study because few tools exist to manipulate this process. Some computational approaches can directly study the permeation of individual ions, but these measurements are often limited to just a few millionths of a second and even the most sensitive of electrical recordings are unable to directly measure ion channel activity on this timescale. Consequently, it is difficult to relate the movement of ions to the 3D structure of a channel and new approaches to this problem are required.Luckily, we have now identified a new class of drugs which interact with the filter gate to directly activate the flow of K+. Interestingly, these drugs work across a range of different K+ channels and therefore provide a new set of tools to manipulate the filter gate and dissect its mechanism of action.In this project we propose to exploit these exciting new tools to manipulate ion permeation through single ion channels. Other approaches will also allow us to compare our measurements with structural and computational models of permeation. In addition, these studies may allow us to classify (and predict) how other K+ channel drugs interact with the channel, and where they may bind. This will have important implications for the rational design of new drugs that target these channels.Overall, this proposal is supported by extensive data that demonstrate the feasibility of our approach. It therefore offers a unique and timely opportunity to understand this most fundamental of biological signalling processes, and it will improve our ability to design new drugs that target K+ channels.