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，这是促进神经细胞与另一个细胞之间通信的分子机器。同样重要的是，这些离子通道是许多治疗药物的靶点，从镇静剂、麻醉剂到抗惊厥药，它们与包括阿尔茨海默病、帕金森氏病以及癫痫在内的一系列毁灭性疾病有关。神经递质门控离子通道的两个主要和重要家族是ATP敏感的P2X受体和五聚体Cys-loop受体，后者包含一个超级受体家族，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。不幸的是，没有高分辨率，原子视图的任何这些受体复合物与其同源神经递质或激动剂。此外，就Cys- loop受体而言，甚至还没有真核受体的高分辨率结构，也没有发表的适合高分辨率结构研究的受体生产方法。如果没有这些信息，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，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整体膜蛋白，它们特别难以分离和结晶。本研究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，设计和实现新的结晶筛选条件，并将这些方法应用于P2X和Cys-loop受体的研究。最具体地说，我们的目标是解决P2X和Cys-loop受体与其同源神经递质和竞争拮抗剂结合的高分辨率x射线晶体结构，通过位点定向诱变和配体结合试验测试定位位点的准确性，并开发这些受体中激动剂和拮抗剂作用的分子机制。综上所述，我们的研究将首次提供这些关键受体在活性和抑制状态下的原子分辨率视图，从而不仅为其生物学功能提供基本见解，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.