Structural biology of neurotransmitter ion channels

描述（由申请人提供）：人类神经系统的正常发育和功能严重依赖于神经递质或配体门控离子通道的功能，这些离子通道是促进神经细胞之间交流的分子机器。同样重要的是，这些离子通道是从镇静剂和麻醉剂到抗惊厥剂的许多治疗剂的靶标，并且它们与包括阿尔茨海默病和帕金森病以及癫痫在内的广泛的破坏性疾病有关或相关。神经递质门控离子通道的两个主要且重要的家族是ATP敏感性P2 X受体和五聚体Cys环受体，后者包括受体的超家族，其包括3-氨基丁酸（GABA）、甘氨酸、血清素和无脊椎动物中的谷氨酸的受体。不幸的是，没有高分辨率，原子视图的任何这些受体的复合物与他们的同源神经递质或激动剂。此外，在Cys-环受体的情况下，甚至还没有真核受体的高分辨率结构或适于高分辨率结构研究的用于产生受体的公开方法.没有这些信息，我们就无法理解这些重要的神经递质门控离子通道是如何“工作”的，最重要的是，我们一方面没有神经递质结合位点的分子图谱，另一方面也没有抑制神经递质活性的分子，即拮抗剂。由于神经递质门控离子通道是多聚体膜蛋白，它们特别难以分离和结晶。本申请中提出的工作的目的是开发新的方法来筛选和制备用于晶体学研究的神经递质门控离子通道，设计和实施新的结晶筛选条件，并将这些方法应用于研究P2 X和Cys环受体。最具体地说，我们的目标是解决高分辨率的X-射线晶体结构的P2 X和半胱氨酸环受体结合到其同源神经递质和竞争性拮抗剂，测试的准确性的定位位点的定点诱变和配体结合试验，并制定分子机制的激动剂和拮抗剂在这些受体的作用。总之，我们的研究将提供这些关键受体在其活性和抑制状态下的第一个原子分辨率视图，从而不仅提供对其生物学功能的基本见解，而且为合理设计新的治疗药物奠定基础。

DESCRIPTION (provided by applicant): The normal development and function of the human nervous system is critically dependent upon the function of neurotransmitter- or ligand-gated ion channels, molecular machines which facilitate the communication between one nerve cell and another. Equally important, these ion channels are the targets of many therapeutic agents, from sedatives and anesthetics to anticonvulsants, and they are implicated or associated with a broad range of devastating diseases including Alzheimer's and Parkinson's diseases as well as epilepsy. Two major and important families of neurotransmitter-gated ion channels are the ATP- sensitive P2X receptors and the pentameric Cys-loop receptors, the latter of which subsumes a super family of receptors that includes those for 3-amino butyric acid (GABA), glycine, serotonin and, in invertebrates, for glutamate. Unfortunately there are no high resolution, atomic views of any of these receptors in complexes with their cognate neurotransmitters or agonists. Furthermore, in the case of Cys- loop receptors, there is not yet even a high resolution structure of a eukaryotic receptor or a published method for production of receptor suitable for high resolution structural studies. Without this information, we are unable to understand how these important neurotransmitter-gated ion channels 'work' and, most significantly, we do not have molecular maps of the binding sites for neurotransmitters on the one hand, and molecules that inhibit neurotransmitter activity, i.e. antagonists, on the other hand. Because neurotransmitter-gated ion channels are multimeric integral membrane proteins they are particularly difficult to isolate and crystallize. The aim of the work proposed in this application is to develop new methods to screen and prepare neurotransmitter-gated ion channels for crystallographic studies, to design and implement new crystallization screening conditions, and to apply these methods to study P2X and Cys-loop receptors. Most specifically, we aim to solve high resolution x-ray crystal structures of P2X and Cys-loop receptors bound to their cognate neurotransmitter and to competitive antagonists, to test the veracity of the mapped sites by site-directed mutagenesis and ligand-binding assays, and to develop molecular mechanisms for the action of agonists and antagonists in these receptors. Taken together, our studies will provide the first atomic resolution views of these crucial receptors in their active and inhibited states, thus not only providing fundamental insight into their biological function, but also laying the foundation for the rational design of new therapeutic agents.