Probing the dynamics of agonist drug interaction with Cys-loop channels by single-molecule recording

离子通道是一种蛋白质，充当“纳米开关”，将电压或化学刺激转化为电流。它们对许多身体功能都至关重要，包括神经元的信息处理和大脑外突触的细胞间交流，这些突触允许神经冲动运动肌肉，调节血压和心率。不出所料，遗传通道突变可以导致人类疾病，从囊性纤维化到神经系统疾病。通道是许多药物的靶点。我们研究的尼古丁类通道介导了安眠药、癫痫药物、尼古丁、酒精、杀虫剂和抗寄生虫药物的作用。如果我们能更好地设计药物来激活或调节尼古丁通道功能，它们可能对其他难以治疗的疾病有用，从慢性疼痛到中风后的痉挛，特别是如果我们能利用通道亚型的巨大多样性来设计对单一亚型有选择性的药物。直到今天，药物都是通过对化学物质的大规模筛选来发现它们是否对人体药物受体有效。这个过程既昂贵又浪费，而且每年很少有新药问世。相反，理想的情况是能够设计出对特定人体通道有特定作用的化学物质。为此，我们需要了解两个步骤：化学物质是如何与通道结合的，以及它是如何改变通道蛋白的形状和功能的。部分问题在于，作为一个开关，通道本身会改变形状，我们不知道这是如何影响药物的结合的。这就是我们想知道的。在伦敦大学学院，我们完善了一项技术，可以观察和解释一个通道蛋白产生的微小电流（比水壶里的电流小10亿倍以上）。这个分析是唯一能告诉我们药物与蛋白质的不同状态结合的紧密程度以及蛋白质在这些不同状态之间移动的速度，不管有没有药物。我们将在我们的工作中使用甘氨酸通道作为尼古丁家族的模型。这个通道具有单分子记录的理想特性，并且最近让我们看到了为什么一些药物在激活受体方面不如其他药物有效，我们发现这个结果也适用于其他尼古丁通道。我们将扩展我们的工作，并以系统的方式获得激活受体的化学物质的这些测量值，并且它们的化学结构彼此不同。我们也将改变蛋白质本身，通过突变适当的位置。将这些信息结合起来，我们就能看到药物与蛋白质最接近的地方。甘氨酸通道也是哺乳动物通道，最接近分辨率较高的x射线结构（无脊椎动物通道，GluCl，发表于2011年6月）。这使得使用与通道函数相关的结构数据成为可能。在牛津大学，我们将通过计算机计算通过与葡萄糖的同源性来模拟甘氨酸通道的结构，并利用这项工作来计划和解释通道功能的实验，以通道的三维形状。最终，我们的工作将引导我们了解化学物质的哪些特征决定了它对尼古丁通道的亲和力和有效性，以及通道的不同部分如何随着激活而移动。它应该告诉我们应该如何修改药物的结构，以使它们更有效。因此，这项基础研究将有助于为未来的药物开发奠定基础，并有望在甘氨酸受体本身（治疗孤儿）和整个尼古丁超家族中进行合理的药物设计。我们的结果也将帮助我们的制药行业同事解释他们的数据，这些数据来自于高通量筛选化合物库中使用的快速分析技术，如结合和宏观功能测量。

Ion channels are proteins that act as "nanoswitches" to translate voltage or chemical stimuli into electrical currents. They are essential to many bodily functions, including information processing in neurones and cell-to cell communication at synapses outside the brain which allow nerve impulses to move muscles and regulate blood pressure and heart rate. Unsurprisingly, inherited channel mutations can produce human disease, from cystic fibrosis to neurological conditions. Channels are targeted by many drugs. The nicotinic-type channels we work on mediate the effects of sleeping pills, drugs for epilepsy, nicotine, alcohol, insecticides and antiparasitic drugs. If we were better at designing drugs to activate or modulate nicotinic channel function, they could be useful in other hard-to-treat conditions, from chronic pain to spasticity after stroke, especially if we could exploit the great diversity of channel subtypes to design agents selective for single subtypes. To this day, drugs are discovered by large scale screening of chemicals to find if they are effective on a human drug receptor. The process is expensive and wasteful, and few new drugs become available every year. Instead of that, it would be ideal to be able to design chemicals to have a specific action on a particular human channel. For this we need to understand two steps: how a chemical binds to the channel and how it then changes the shape and function of the channel protein. Part of the problem is that, working as a switch, the channel itself changes shape and we don't know how this affects the binding of the drug.This is what we want to find out. At UCL we perfected a technique to see and interpret the tiny current (more than a billion times smaller than the current in a kettle) produced by one channel protein. This analysis is the only one that can tell us how tightly the drug binds to different states of the protein and how quickly the protein moves between these different states, with and without the drug. We will use in our work the glycine channel as a model for the nicotinic family. This channel has ideal properties for single molecule recording and has recently allowed us to see why some drugs are less effective than others in turning the receptor on, a result we found to be applicable to other nicotinic channels. We will extend our work and obtain these measurements for chemicals that activate the receptor, and differ from each other in their chemical structure in a systematic way. We will also change the protein itself, by mutating appropriate positions. Combining this information will allow us to see where the drugs "touch" the protein most closely. Glycine channels are also the mammalian channel that is closest to a well-resolved X-ray structure (that of an invertebrate channel, GluCl, published in June 2011). This makes it possible to use the structural data in relation to channel function. At Oxford we will model the structure of the glycine channel by homology to GluCl by computer calculations and use this work to plan and interpret the experiments on channel function in terms of channel 3-D shape. Ultimately our work should lead us to understand what features in a chemical determine its affinity and efficacy for a nicotinic channel and how the different parts of the channel move with activation. It should give us indications on how the structure of drugs should be modified, in order to make them more effective. Hence this fundamental research will be useful to lay the basis for future drug development and hopefully enable rational drug design, in the glycine receptor itself (a therapeutic orphan) and in the nicotinic superfamily as a whole. Our results will also help our drug industry colleagues interpret their data that come from the quick assay techniques used by in high-throughput screening of libraries of compounds, such as binding and macroscopic functional measurements.