Identifying the Interactions between Animal Toxins and Human nAChRs: The Role of Snake PLA2 in Interacting with nAChR alpha Subunits

项目总结/摘要 烟碱型乙酰胆碱受体（nAChR）参与多种基本生理过程， 这些受体的功能障碍与许多人类疾病有关，如阿尔茨海默氏症， 帕金森氏症。人nAChR的结构和功能被广泛研究;然而， 不同nAChR亚基的组合，形成各种nAChR亚型及其胞外区 结合一系列结构多样的配体，在高通量（HT）和系统性方面提出了挑战。 nAChRs的研究。幸运的是，动物毒素提供了一个独特的工具来研究的结构和功能， 因为它们已经通过数百万年的自然选择进行了微调， 对特定蛋白质如nAChR的特异性和选择性。我们的长期目标是确定普遍的 或与人类nAChR相互作用的选择性动物毒素，并阐明其机制基础 在他们的互动中。为了实现这一目标，本申请的总体目标是建立HT 确定蛇毒素和nAChRs亚基之间相互作用的方法，以及破译 毒素在与人类nAChR相互作用中的作用，提出以下三个具体目标：1） 生成蛇毒素ORFeome文库。独特的蛇毒转录本开放阅读框（ORF）将 从医学上重要的蛇（Crotalus atrox）的现有毒腺cDNA文库中鉴定，和 单独地连接到“供体”载体中以产生蛇毒素ORFeome文库，其可直接用于 与包括nAChR在内的任何人类蛋白质的相互作用检测。2)确定蛇的相互作用 毒素与人类nAChR亚单位。蛇毒素和人类nAChR亚基之间的相互作用， 重点是毒液磷脂酶A2（PLA 2）和nAChR α亚基之间的检测，将通过 利用HT酵母双杂交（HT-Y2 H）系统，随后进行共亲和纯化/质谱分析 (AP/MS）。3)破译毒液毒素与nAChR亚基相互作用的作用。我们将开发一个HT 药理学测定以确定蛇毒素如PLA 2是否作为抗人的激动剂或拮抗剂 nAChRs。将nAChR如α1β1γδ、α3β4、α4β2和α7单独转染到人细胞中 例如HEK 293，用于测量由PLA 2-nAChR引起的钙动力学和膜电位 交互.研究结果预计将产生影响，因为：1）它将提供宝贵的资源 为科学界、大学教师和学生提供后续生物学研究和生物医学 应用; 2）它将为HT和结构的系统研究提供独特的工具， nAChRs的功能。此外，该项目将为本科生创造许多机会， 提高他们在生物医学科学方面的研究技能。

PROJECT SUMMARY/ABSTRACT Nicotinic acetylcholine receptors (nAChRs) are involved in a variety of fundamental physiological processes, and dysfunction in these receptors is associated with many human disorders such as Alzheimer’s and Parkinson’s diseases. The structure and function of human nAChRs are extensively studied; however, the combinations of different nAChR subunits making various nAChR subtypes and their extracellular regions binding a structurally diverse range of ligands, pose challenges in high-throughput (HT) and systematic investigation of nAChRs. Fortunately, animal toxins provide a unique tool to study the structure and function of nAChR because they have been fine-tuned through millions of years of natural selection to present high specificity and selectivity towards specific proteins such as nAChRs. Our long-term goal is to identify universal or selective animal toxins that interact with human nAChRs, and elucidate the mechanistic underpinnings underlying their interactions. To achieve this goal, the overall objective of this application is to establish HT approaches for identifying the interactions between snake toxins and nAChRs subunits, as well as deciphering the role of toxins in interacting with human nAChRs by proposing the following three specific aims: 1) Generate a snake toxin ORFeome library. The unique snake toxin transcript open-reading frames (ORFs) will be identified from the existing venom gland cDNA library of a medically important snake (Crotalus atrox), and individually ligated into a “donor” vector to create a snake toxin ORFeome library that can be directly used for the interaction detections with any human proteins including nAChRs. 2) Determine the interactions of snake toxins with human nAChR subunits. The interactions between snake toxins and human nAChR subunits, with focus on the detection between venom phospholipase A2 (PLA2) and nAChR α subunits, will be identified by utilizing a HT yeast two-hybrid (HT-Y2H) system, followed by co-affinity purification/mass spectrometry (AP/MS). 3) Decipher the role of venom toxins in interacting with nAChR subunits. We will develop a HT pharmacological assay to determine if snake toxins such as PLA2 act as agonist or antagonist against human nAChRs. The nAChRs such as α1β1γδ, α3β4, α4β2, and α7, will be individually transfected into human cells such as HEK293 for measuring the calcium dynamics and membrane potential elicited by PLA2-nAChR interactions. The research results are expected to be impactful because: 1) it will provide valuable resources for the scientific community, university faculty and students for subsequent biological research and biomedical applications; and 2) it will provide a unique tool for HT and systematic investigation of the structure and function of nAChRs. In addition, the project will create numerous opportunities for undergraduate students to improve their research skills in biomedical science.