Combining structure and function in the nicotinic superfamily: the single-channel activation mechanism for the prokaryotic model channel ELIC

我们工作的目的是了解离子通道如何作为分子发挥作用。关键是要知道通道的三维结构（通过x射线晶体学），并通过记录其电活动来找出通道激活时该结构是如何移动的。对于同一分子，几乎不可能同时获得这两种信息。新的数据意味着这对于我们正在研究的频道组来说是可能的。离子通道是我们基因组中编码的蛋白质，是我们体内许多细胞的重要组成部分。例如，它们允许细胞在称为突触的细胞间连接处相互交流。这不仅对大脑很重要，而且对我们身体的其他部分也很重要，因为它允许适当的命令到达我们四肢的肌肉，调节我们的血压和心率。通道还允许每个神经元处理从其他神经元接收到的信息。经络对人体正常生理和疾病都很重要。编码通道的基因突变会破坏通道功能，并产生遗传性人类疾病，如囊性纤维化。除此之外，许多用于常见病或麻醉的药物都是通过与通道结合而起作用的。特别是，我们研究的一组通道，尼古丁超家族，是安眠药、癫痫药物、烟草中的尼古丁和一些杀虫剂的目标。关于通道的信息主要有两个来源：第一个是x射线晶体学，它为我们提供有关蛋白质形状的信息；第二个是电生理学，它测量通道产生的电信号。在最先进的形式中，这是我们的特殊专长，这项技术可以实时检测通过单个蛋白质分子的电流，即使它非常小（比水壶里的电流小10亿倍以上）。这项技术非常有用，因为它使我们能够测量通道功能中分子事件发生的速度。因此，我们可以把通道的功能精确地理解为一种化学反应，量化从神经递质结合到通道打开的每一步。通过在烟碱组的通道中这样做，我们发现了当蛋白质活跃或不活跃时，神经递质和药物与蛋白质的结合有多紧密，以及为什么有些药物的作用比其他药物更强。理想情况下，我们应该研究同信道的结构和功能。这并不容易，因为通道很难结晶，到目前为止，我们在这类通道中只有三个通道（GLIC， ELIC和GluCl）具有良好的结构。其中，GLIC产生的电信号太小，不利于良好的电生理。至于葡萄糖，我们不知道它的信号有多好（这个结构刚刚被发表）。葡萄糖的一个潜在问题是，它不像其他通道一样打开，对神经递质类化合物做出反应，但它需要两种不同的物质，结合在不同的地方，所以我们不知道它会是一个多好的模型。直到现在，ELIC被认为是无法打开的。其他科学家现在已经发现了激活ELIC的合适物质，结果证明ELIC打开良好，并发出良好的大信号。我们想把我们的专长——单分子记录技术应用到ELIC上，这样我们就能理解它作为一个分子是如何起作用的。一旦我们掌握了这一点，我们就可以进一步推动我们的理解，因为我们可以参考精确的结构信息（可用于ELIC）来解释药物的作用和通道中突变的影响。这是基础研究，但如果我们想要解释分子的哪些部分发生了变化，当通道被激活时它们是如何移动的，药物与蛋白质结合的确切位置，以及我们应该如何修改药物结构以使它们更有效，这是必要的。

The purpose of our work is to understand how ion channels function as molecules. The key to this is to know the 3-D structure of the channel (by X-ray crystallography) and to find out how this structure moves when the channel is activated, mainly by recording its electrical activity. It is rarely possible to get both sorts of information for the same molecule. New data mean that this is now possible for the group of channels we work on.Ion channels are proteins coded in our genome and are essential components of many cells in our bodies. For instance, they allow cells to communicate with each other at cell-to-cell junctions called synapses. This is essential not only in the brain but also to in the rest of our bodies where it allows the appropriate commands to reach muscles in our limbs and to regulate our blood pressure and heart rate. Channels also allow each neurone to process the information it receives from other neurones. Channels are important for normal human physiology and for disease. Mutations in the genes that code for channels can damage channel function and produce inherited human disease, such as cystic fibrosis. In addition to that, many drugs used for common diseases or in anaesthesia act by binding to channels. In particular, the group of channels that we study, the nicotinic superfamily, are targeted by sleeping pills, drugs for epilepsy, nicotine in tobacco and some insecticides. There are two main sources of information about channels: the first is X-ray crystallography, which provides us with information about the shape of the protein and the second is electrophysiology, which measures the electrical signal the channel produces. In the most advanced form, which is our special expertise, this technique detects the current that passes through a single protein molecule in real time, even though it is very small (more than a billion times smaller than the current in a kettle). This technique is very useful, because it allows us to measure the speed with which molecular events in the function of the channel occur. Hence we can understand channel function precisely as a chemical reaction, quantifying each step, from the binding of the neurotransmitter to the opening of the channel. By doing this in channels in the nicotinic group, we have found how tightly neurotransmitters and drugs bind to the protein when it is active or inactive and why some drugs act more strongly than others. Ideally we should study the structure and the function of the SAME channel. This is not easy because channels are difficult to crystallize, and so far we have good structures only for three channels in this group (GLIC, ELIC and GluCl). Of these, GLIC produces electrical signals that are too small for good electrophysiology. As for GluCl, we don't know how good its signal is (the structure has literally just been published). A potential problem with GluCl is that it does not open like all other channels in the group do, in response to a neurotransmitter-like compound, but it requires TWO different substances, binding to different places, so we don't know how good a model it will be.Until now ELIC was thought not to be able to open. Other scientists have now discovered the right substances that activate ELIC, and it turns out to open well and to give an excellent, big signal. We want to apply the single-molecule recording that is our special skill to ELIC, so that we can understand how it functions as a molecule. Once we have that, we can push our understanding much further, because we can refer to precise structural information (available for ELIC) in interpreting the effect of drugs and the effect of mutations in the channel. This is basic research but it is what is needed if we want to explain what bits of the molecule change and how they move when the channel is activated, where exactly drugs bind to the protein and how we should modify the structure of drugs in order to make them more effective.