Structural analysis of human GABAA receptors

人脑是地球上最强大的生物处理器。它也是极其复杂的。它包含大约1000亿个脑细胞，以及这些脑细胞之间超过1万亿个连接，这些连接引导着大量信息的流动。显然，如此复杂的制度要想顺利运作，就需要规则。这需要一个监管体系。而我们研究的靶点--γ-氨基丁酸A型受体（GABA-A-Rs）是该调节系统中最重要的组成部分，是表达于神经细胞表面的蛋白质。它们存在于整个大脑中，允许神经细胞对神经递质γ-氨基丁酸（称为GABA）做出反应。神经递质GABA作用于GABA-A-R，向神经细胞发送停止信号。这意味着GABA-A-R是大脑的刹车。他们传播平静。它们调节兴奋。因此，毫不奇怪，当这些调节器失灵时，结果是大脑活动加剧，导致一系列使人衰弱的疾病，如失眠、焦虑症和癫痫症，这些疾病影响着全世界数千万人。幸运的是，有可用的治疗方法，最著名的是苯二氮卓类药物，它可以加强GABA-A-Rs的镇静作用。不幸的是，由于GABA-A-R对大脑中的许多不同过程至关重要，因此苯二氮卓类药物具有副作用。此外，GABA-A-R在大脑功能中发挥的许多作用还远未完全解决。例如，GABA-A-R也是全身麻醉剂和大脑自身产生的抗应激分子（神经类固醇）的靶点。它们也是抑郁症和慢性疼痛的靶点，并正在研究作为精神分裂症，中风和酒精成瘾的治疗药物。因此，为了更好地理解GABA-A-R如何促进大脑功能，尽可能全面地了解这些蛋白质是如何运作的是至关重要的。以高（原子或近原子）分辨率获得GABA-A-Rs的三维地图提供了重要的信息，例如，这些生物机器如何对神经递质GABA做出反应，以内部重组自己，打开一个内置的门，让离子流过神经细胞的膜。这些图谱还将揭示GABA-A-R如何结合人脑中的临床相关药物和天然配体。最后，人脑中存在不同的GABA-A-R群体，每个群体具有独特的结构、独特的功能、独特的表达谱和独特的药理学。因此，获得每个不同GABA-A-Rs群体的结构数据将揭示它们的区别特征以及这些特征如何赋予独特的性质。此外，详细的结构允许可视化与GABA-A-R结合的药物，这将为未来改进的治疗方法的设计提供信息。需要大量稳定形式的高纯度GABA-A-R，这是特别困难的，因为GABA-A-R是非常脆弱的蛋白质。幸运的是，在我们的实验室里，我们拥有最先进的蛋白质生产、纯化、筛选和结构测定技术。这就是为什么今年（2014年）我们成功地发表了GABA-A-R的第一个高分辨率结构，揭示了它的运作模式和它所包含的药物结合腔的数据。然而，一个单一的结构只是一个快照。为了更好地了解蛋白质的功能，需要这些生物机器以及其他亚型的许多替代结构（快照）。我们相信，通过在未来获得更多的GABA-A-R结构，我们将大大提高对GABA-A-R如何影响大脑功能的理解，并告知制药企业如何设计改进的治疗方法。

The human brain is the most powerful biological processor on the planet. It is also extremely complicated. It contains around one-hundred billion brain cells and over a trillion connections between those brain cells which channel the flow of vast quantities of information. Clearly such a complicated system requires rules if it is to operate smoothly. It requires a regulatory system. And the targets of our research, gamma-aminobutyric acid Type-A receptors, termed GABA-A-Rs, are a most important part of this regulatory system.GABA-A-Rs are proteins expressed at the surface of nerve cells. They exist throughout the brain to allow nerve cells to respond to the neurotransmitter gamma-aminobutyric acid, called GABA. The neurotransmitter GABA acts upon GABA-A-Rs to send a stop signal to nerve cells. This means GABA-A-Rs are the brakes on the brain. They spread calm. They regulate excitement. And so it is no surprise that when these regulators fail the result is heightened brain activity, which leads to a range of debilitating illnesses such as insomnia, anxiety-disorders and epilepsy, disorders that affect tens of millions of people worldwide. Fortunately there are treatments available, most famously benzodiazepines which reinforce the calming influence of GABA-A-Rs. Unfortunately, because GABA-A-Rs are vital to so many different processes in the brain benzodiazepines have side-effects. Furthermore, the many roles that GABA-A-Rs play in brain function are far from fully resolved. For example, GABA-A-Rs are also targets for general anaesthetics and for the brains home-grown anti-stress molecules, the neurosteroids. They are also targets in depression and chronic pain, and are being investigated as therapeutics in schizophrenia, stroke and alcohol addiction. Thus, it is vital to understand as fully as possible how these proteins operate in order to better understand how GABA-A-Rs contribute to brain function.One way to do this is to analyse the structures of GABA-A-Rs. Obtaining three-dimensional maps of GABA-A-Rs at high (atomic or near-atomic) resolution provides vital information on, for example, how these biological machines respond to the neurotransmitter GABA to internally reorganisation themselves to open a built-in gate that lets ions flow across the membranes of nerve cells. Such maps will also reveal how GABA-A-Rs bind clinically relevant drugs and natural ligands in the human brain. Finally, there are different populations of GABA-A-Rs in the human brain, with each population having unique structures, unique functions, unique expression profiles, and unique pharmacology. So obtaining structural data for each of the distinct GABA-A-Rs populations will unveil their distinguishing features and how these features imbue unique properties. Furthermore, detailed structures permit visualisation of drugs bound to GABA-A-Rs, which will inform on design of improved therapeutics in the future.However, obtaining high-resolution structures of GABA-A-Rs is extremely challenging. Large quantities of highly pure GABA-A-Rs in a stable form are required, something that is especially difficult because GABA-A-Rs are very delicate proteins. Fortunately in our laboratory we have state-of-the-art technologies for protein production, purification, screening, and structure determination. That is why this year (2014) we were successful in publishing the first ever high resolution structure of a GABA-A-R, revealing data on its mode of operation and on the drug-binding cavities it contains. However, a single structure is only a snap-shot. To gain a better understanding of protein function many alternative structures (snap shots) of these biological machines in action are required, as well as of other subtypes. We believe that by obtaining more GABA-A-R structures in the future we will substantially enhance the understanding of how GABA-A-Rs impact on brain function and inform pharmaceutical enterprise on how to design improved therapeutics.