Structural studies into human muscle nicotinic acetylcholine receptors

肌肉型烟碱乙酰胆碱受体（Muscle type nicotinic acetylcholine receptor，nAChR）是神经肌肉接头的关键组成部分，它接受来自运动神经元的乙酰胆碱信号，启动肌肉膜去极化和肌肉收缩。它们是五聚体配体门控离子通道家族的原型成员，该家族还包括其他神经递质如GABA、血清素和甘氨酸的受体。这些受体的早期发现帮助开发了许多用于研究离子通道的方法，这导致了我们对离子通道功能的理解。肌肉型nAChR也是五聚体配体门控离子通道家族中最复杂的成员之一，由4个不同的亚基组成- 2 α 1，1 β 1，1 δ和胎儿受体中的γ亚基，或者是成人受体中的一个α亚基。成人受体的表达在出生前几周开始，之后它被选择性地并入神经肌肉接头。这种受体的缺失导致肌无力综合征，其中患者可能患有不同严重程度的肌无力，从胎儿运动不能和子宫内死亡到导致轻度终身残疾的疲劳性肌无力。到目前为止，关于人体肌肉nAChR受体结构的信息很少，大多数研究都是在电射线的电器官受体上进行的。虽然这种直系同源物与人类胎儿nAChR具有相似的亚基组成，但它并不能完全概括人类蛋白质的行为，并且不能可靠地用于预测在患者中鉴定的遗传变异是否会导致疾病。这是因为致病的变异体最常见于γ亚基，而γ亚基不存在于电射线中。这些直向同源物的结构也不够准确的药物发现，其中基于结构的方法大大加快了新药的开发，本提案的目的是通过在近原子分辨率下解析蛋白质的结构，然后详细评估蛋白质如何工作，从而获得人类肌肉型nAChRs如何工作的详细图片。我们已经建立了成人和胎儿受体的蛋白质生产和纯化方法，我们用它来产生一个有前途的初步冷冻电子显微镜数据集的成人受体在9 μ m分辨率。为了产生更高分辨率的结构信息，我们将改进这些方法，并应用各种其他策略将其捕获在不同的功能状态，包括使用致病的受体变体和使用已知靶向受体的药物。在这些结果的基础上，我们将研究结构导向突变的功能和对不同刺激的反应。这些将破译通道的结构-功能关系和它如何工作的分子机制。这些信息最终将有助于预测在患者中观察到的遗传变化是否可能导致疾病，并有助于设计针对这种蛋白质的新药来治疗肌无力和其他形式的神经肌肉疾病。

Muscle type nicotinic acetylcholine receptors (nAChR) are the key components of the neuromuscular junction that receive the acetylcholine signal from the motor neurone to initiate muscle membrane depolarisation and muscle contraction. They are the archetypal members of the pentameric ligand-gated ion channel family that also includes receptors to other neurotransmitters such as GABA, serotonin, and glycine. The early discovery of these receptors helped develop many of the methods used to study ion channels, which led to much of our understanding of how ion channels function.Muscle type nAChR are also some of the most complex members of the pentameric ligand-gated ion channel family and is made up of 4 different subunits - 2 alpha1, 1 beta1, 1 delta and either a gamma subunit in foetal receptors, or an epsilon subunit in adult receptors. Expression of the adult receptor initiates a few weeks before birth, after which it is selectively incorporated into the neuromuscular junction. Abnormalities in this receptor lead to myasthenic syndromes where patients can suffer from muscle weakness of varying severity from foetal akinesia and death in utero to fatigable muscle weakness that causes a mild lifelong disability. So far, very little information is available on the structure of the human muscle nAChR receptors, with most of the studies carried out on the electric organ receptor of the electric ray. While this ortholog has similar subunit composition to human foetal nAChR, it nevertheless does not fully recapitulate the human protein's behaviour and cannot be reliably used to predict whether genetic variants identified in patients cause disease. This is because disease causing variants are most commonly found in the epsilon subunit, which is not present in the electric ray. These orthologue structures are also not accurate enough for drug discovery, where structure based approaches greatly accelerate the development of new medicines.The aim of this proposal is to obtain a detailed picture of how human muscle type nAChRs work by resolving structures of the proteins at near atomic resolution, followed by detailed assessments of how the protein works. We have already established protein production and purification methods for both adult and foetal receptors, which we used to produce a promising preliminary cryo-electron microscopy dataset of the adult receptor at 9 Å resolution. To produce higher-resolution structural information, we will refine these methods and apply a variety of other strategies to trap it in different functional states, including using disease-causing variants of the receptor and using drugs that are known to target the receptor.Building on these results, we will examine the function and responses to different stimuli with structure-guided mutations. These will decipher the channel's structure-function relationship and the molecular mechanisms that underlie how it works. This information will ultimately help predict whether genetic changes observed in patients are likely to cause disease, as well as assist in designing new drugs that target this protein to treat myasthenia and other forms of neuromuscular diseases.