STRUCTURAL AND FUNCTIONAL FEATURES OF THE 5-HT3R BINDING SITE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。研究进展：特定目的：开发与5-HT3R具有相同或相似配体特异性的3型血清素受体的可溶性氨基末端结合域。本项目于2004年5月1日启动，第一年目标如下：建立PIs实验室。2. 聘请2名研究生和一名研究助理。建立稳定表达乙酰胆碱结合蛋白（AChBP）的细胞系。第一年的所有目标都已完成。在过去的12个月里，有两名研究生被聘用。一名学生于2004年8月开始工作，另一名学生于2005年1月开始工作。两个学生都在做这个项目。2005年3月聘请了一名研究助理（Jestina Kusina博士），并开始在实验室开展主要侧重于方法开发的工作。所有实验室设备都已购买，目前正在项目中使用。活跃的研究开始于2004年11月，主要集中在发展表达系统和提案中概述的蛋白质纯化协议。我们已经开发出表达AChBP的稳定转染细胞系。该蛋白的纯化和特性将很快开始。AChBP的药理学将通过96孔分析和表面等离子体共振（SPR）进行表征。Kusina博士目前正在为此目的开发SPR检测方法。AChBP/5-HT3R嵌合体的合成目前正在进行中，应该在我们制定支持协议时完成。项目目标已略微扩大，包括使用表达的AChBP作为药物开发中的生物传感器。这将使我们能够利用AChBP表达系统和SPR来评估该蛋白的药理学特异性。由于AChBP被用作许多其他配体门控离子通道的结构模板，这些数据应该对LGIC领域做出有价值的贡献。我们也在探索使用AChBP/SPR传感器检测重症肌无力患者的抗nachr抗体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Progress: Specific Aim: Develop a soluble amino terminal binding domain of the serotonin type 3 receptor with ligand specificities identical or similar to the 5-HT3R. This project began May 1, 2004 with the following first year goals: 1. Establish the PIs laboratory. 2. Hire 2 graduate students and a research Associate 3. Establish stably expressing cell lines for the Acetylcholine Binding Protein (AChBP). All first year goals have been accomplished. Two graduate students were hired over the past 12 months. One student began working in August 2004 and a second in January 2005. Both students are working on this project. A Research Associate (Dr. Jestina Kusina) was hired in March 2005 and has begun work in the laboratory primarily focusing on methods development. All laboratory equipment has been purchased and is currently being employed in the project. Active research began in November 2004 and has been focused on developing the expression systems and protein purification protocols outlined in the proposal. We have developed stably transfected cells lines expressing the AChBP. The purification and characterization of this protein will begin shortly. AChBP pharmacology will be characterized by 96 well assays and Surface Plasmon Resonance (SPR). Dr. Kusina is currently developing the SPR assays for this purpose. Syntheses of the AChBP/5-HT3R chimeras are currently underway and should be complete by the time we have developed the supporting protocols. Project goals have been expanded slightly to include the use of the expressed AChBP as a biosensor in drug development. This will enable us to make use of the AChBP expression system and SPR to evaluate the pharmacological specificity of this protein. Since the AChBP is being used as a structural template for many other ligand-gated ion channels, these data should constitute a valuable contribution to the LGIC field. We are also exploring the use of an AChBP/SPR sensor for detection of anti-nAChR antibodies in Myasthenia Gravis.