STRUCTURAL AND FUNCTIONAL FEATURES OF THE 5-HT3R BINDING SITE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。进展：特定目的：开发5-羟色胺3型受体的可溶氨基末端结合域，其配体特异性与5-HT3R相同或相似。该项目于2004年5月1日开始，第一年的目标如下：1.建立PIS实验室。2.聘请2名研究生和1名研究助理3.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，雇佣了两名研究生。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月聘用了一名研究助理(Jestina Kusina博士)，并已开始在实验室开展工作，主要侧重于方法开发。所有实验室设备都已采购完毕，目前正在该项目中使用。积极的研究始于2004年11月，一直专注于开发提案中概述的表达系统和蛋白质纯化方案。我们已经建立了稳定表达AChBP的细胞系。该蛋白的提纯和鉴定工作将很快开始。AChBP药理学将通过96个井分析和表面等离子激元共振(SPR)来表征。库西纳博士目前正在为此目的开发SPR分析。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的时候完成。项目目标已略有扩大，包括将表达的AChBP用作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，这些数据应该构成对LGIC领域的宝贵贡献。我们还在探索使用AChBP/SPR传感器来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.