Single molecule quantification of the activation, biophysics and pharmacology of GlyREM, a new structural model for pentameric ligand-gated channels

离子通道存在于我们所有的细胞中。当神经递质或药物与之结合时，我们研究的通道就会打开，并在突触（包括大脑中的突触）上介导细胞间的快速通信。通道在疾病中也很重要：通道基因的突变可导致遗传性人类疾病，如囊性纤维化。此外，许多用于常见病或麻醉的药物通过与通道结合而起作用。例如，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。我们研究的目的是了解离子通道如何作为分子发挥作用。为此，我们需要知道它们的三维结构，以及当通道被激活时这种结构是如何变化的。结构通常通过晶体学获得，功能通过记录通道的电活动获得。对于通道来说，获得可以用于结构的好的晶体是缓慢而困难的。要得到多个结构来显示通道在激活时如何改变形状就更难了。此外，一些可以成像的通道不能提供很好的功能数据，因此结构和功能的整合是困难的。一项不需要晶体的技术的进步，低温电子显微镜（EM）正在改变这一点，并开始给出GlyR-EM的结构，GlyR-EM是我实验室专门研究的一组通道的形式，甘氨酸受体。我们建议在这种形式的渠道上工作，与正在进行结构工作的美国小组协调我们的工作，以获得最大的洞察力。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分子产生的微小电流（比水壶使用的电流小10亿倍）。这项工作是必要的，因为这是测量神经递质和药物在不同形状的通道上结合的紧密程度，以及在有和没有药物的情况下，通道在这些不同状态之间移动的速度的唯一方法。它也是唯一一种可以准确测量药物强度（用药理学家的行话来说，就是药效）的技术，因为它可以测量药物在结合后保持通道畅通的能力。在甘氨酸通道中，对这些数据的分析使我们发现强效药物之所以强效，是因为它们能有效地产生初始构象变化。之后，所有药物的通道都以类似的方式打开。这项研究开始于甘氨酸通道，但现在已知这一发现适用于所有已测试的尼古丁通道。很明显，了解通道结构在初始阶段是如何移动的是很重要的，为此我们需要将我们的功能工作与结构生物学家的工作结合起来，后者可以对通道进行成像。我们的试验数据表明，GlyR-EM通道提供良好的电信号，与我们研究过的各种形式的人类甘氨酸受体略有不同。这不是问题，相反，这些差异为我们提供了决定通道功能属性的信息。对于甘氨酸受体来说尤其如此，不同形式的氨基酸序列非常相似，这使得功能差异的原因可能更容易识别。最后，Cryo-EM是在与我们通常选择的电记录不同的条件下进行的，并且在这些条件下（低温和保持电位，长期药物应用）没有通道功能的信息。我们必须做这些实验，以便我们能够识别和解释新的结构。重要的是，伦敦大学学院的功能工作将使我们能够向我们的美国合作者指出哪些新的结构实验将是最有用和信息丰富的。这是基础研究，但最终它可以为我们提供如何修改药物结构以使其更有效的信息。

Ion channels are present in all our cells. The channels we work on open when a neurotransmitter or a drug binds to them, and mediate fast cell-to-cell communication at synapses, including those in the brain. Channels are also important in disease: mutations in channel genes can cause heritable human disease, such as cystic fibrosis. Also, many drugs used for common diseases or in anaesthesia act by binding to channels. For instance, the group of channels that we study, the nicotinic superfamily, are targeted by sleeping pills, drugs for epilepsy, the nicotine in tobacco and some insecticides. The aim of our research is to understand how ion channels function as molecules. For this we need to know their 3-D structure and how this changes when the channel is activated. Structure is usually obtained by crystallography, and function by recording the channel's electrical activity. For channels, it is slow and difficult to get good crystals that can be used for structure. It is even harder to get multiple structures that show how the channels change shape as they activate. Furthermore, some of the channels that can be imaged do not give good functional data, so integrating structure and function is difficult.Progress in a technique that does not require crystals, cryo electronmicroscopy (EM) is changing this, and is beginning to give structures for GlyR-EM, a form of the very group of channels my lab specialises on, glycine receptors. We propose to work on this form of channel, co-ordinating our work with the US group that is doing the structural work, to obtain the maximum insight.At UCL, we perfected a technique to see and interpret the tiny currents (a billion times smaller than the current used by a kettle) produced by one channel molecule. This work is needed, because it is the only way to measure how tightly neurotransmitters and drugs bind to the channel in its different shapes, and how quickly the channel moves between these different states, with and without the drug. It is also the only technique that can measure accurately how strong (in pharmacologist's jargon, how efficacious) a drug is, because it measures how good the drug is at keeping the channel open, when it has bound. In glycine channels, analysis of these data allowed us to find out that strong drugs are strong because they are effective at producing the initial conformational change. After that, the channel opens in a similar manner for all drugs. This research started on glycine channels, but the finding is now known to apply to all nicotinic channels that have been tested. It is obviously important to understand how the channel structure moves in this initial step and for that we need to integrate our functional work with that of the structural biologists that can image the channel.Our pilot data show that the GlyR-EM channel gives good electrical signals, and is slightly different from the various forms of human glycine receptor we have worked with. This is not a problem, on the contrary, the differences give us information on what determines the functional properties of the channel. This is particularly true for glycine receptors, where the amino-acid sequence of the different forms is very similar, making the causes of the differences in function potentially easier to identify.Finally Cryo-EM is carried out in conditions that are different from the ones we normally would choose for electrical recording, and there is no information on channel function in these conditions (low temperature and holding potential, long drug applications). We must do these experiments so that we can identify and interpret the new structures. Importantly, the UCL functional work will allow us to indicate to our US collaborators which new structural experiments would be the most useful and informative.This is basic research but, ultimately it can give us information on how we should modify the structure of drugs in order to make them more effective.