Structure Function Studies on the Mechanisms of P2X3 Receptor Desensitization

描述（由申请方提供）：嘌呤能受体（P2 X）是通过ATP和腺苷结合激活的蛋白质。由于它们在心血管、神经和免疫系统中积极表达，因此这些受体发挥功能的结构机制对于理解它们如何调节平滑肌收缩、血小板活化、突触传递和炎症等过程非常重要。因此，它们已成为心血管、神经元和炎性疾病的关键潜在药理学靶标。尽管是一个积极追求的药理学目标，只有两个高分辨率的结构存在的任何嘌呤受体亚型，既不是在人类同系物。斑马鱼P2 X4受体的脱辅基封闭状态和ATP结合状态的结构为受体激活提供了一些结构上的见解;然而，脱敏（快速与缓慢）的机制仍然没有答案。由于P2 X3受体显示快速脱敏动力学，而P2 X4显示缓慢脱敏动力学，因此两种受体之间的结构差异将使我们深入了解脱敏机制。该提案概述了使用已证实的策略，以确定在脱敏状态下使用X射线晶体学的人P2 X3受体的高分辨率结构。首先，我将鉴定人P2 X3的“最小功能构建体”，其显示与野生型相同的激活和脱敏性质，但对于在哺乳动物表达系统中的表达和纯化是最佳的。通过这个过程，我将确定关键残基的N-和C-末端结构域的人P2 X3的激活和脱敏是重要的。结晶试验将不限于使用去污剂/蛋白质胶束的经典方法，还将包括允许在富含脂质的环境中结晶的新技术，例如双胶束和无规立方相结晶。为了增加获得减敏状态的结构的可能性，将进行共结晶 在已知稳定脱敏状态的两种毒素以及来自构象特异性抗体的Fab片段的存在下。这项工作的结果将大大有助于嘌呤受体激活和随后的脱敏机制的理解。

DESCRIPTION (provided by applicant): Purinergic receptors (P2X) are proteins that are activated by the binding of ATP and adenosine. Because they are actively expressed in the cardiovascular, nervous, and immune systems, the structural mechanisms by which these receptors function are important to understand how they modulate processes such as smooth muscle contraction, platelet activation, synaptic transmission and inflammation. As such, they have become a key potential pharmacologic target for cardiovascular, neuronal and inflammatory diseases. Despite being an actively pursued pharmacologic target, only two high-resolution structures exist for any of the purinergic receptor subtypes and neither is on a human homolog. Structures of an apo-closed state as well as an ATP-bound state of zebra fish P2X4 receptor have provided some structural insight into receptor activation; however, the mechanisms surrounding desensitization (rapid vs. slow) remain unanswered. Because P2X3 receptor displays rapid desensitization kinetics, as opposed to P2X4, which displays slow desensitization kinetics, structural differences between the two receptors will give insight into mechanisms of desensitization. This proposal outlines the use of proven strategies to determine a high-resolution structure of human P2X3 receptor in the desensitized state using X-ray crystallography. First, I will identify a "minimal functional construct" of human P2X3 which displays activation and desensitization properties identical to wild type yet is optimal for expression and purification in a mammalian expression system. Through this process, I will identify key residues in the N- and C-terminal domains of human P2X3 that are important for activation and desensitization. Crystallization trials will not be limited to classical methods usig detergent/protein micelles but will also include new techniques that allow crystallization in a lipid-rich environment, such as bicelle and lipidic cubic phase crystallization. To increase the likelihood of obtaining a structure of the desensitized state, co-crystallization will be performed in the presence of two toxins that are known to stabilize the desensitized state as well as with Fab fragments from conformation-specific antibodies. The results of this work will substantially contribute to the understanding of the mechanisms for purinergic receptor activation and subsequent desensitization.