CALCIUM INFLUX PATHWAYS IN ALLERGY

过敏症中的钙流入途径

• 批准号: 6534224
• 负责人: JEAN-PIERRE M KINET
• 金额: $ 39.8万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534224
• 关键词: CHO cells; Xenopus oocyte; biological signal transduction; calcium channel; calcium flux; calcium indicator; cell membrane; cellular immunity; electrophysiology; hypersensitivity; immunoprecipitation; mass spectrometry; mast cell; membrane channels; membrane transport proteins; phosphorylation; protein protein interaction; protein structure function; spectrometry; tissue /cell culture; voltage /patch clamp; western blottings; yeast two hybrid system

DESCRIPTION: (Adapted from the applicant's abstract) Sustained calcium entry in mast cells plays a critical role in the initiation and maintenance of allergic responses associated with ligand binding to Fc epsilon RI. It is believed that the sustained calcium entry associated with engagement of Fc epsilon RI is mediated by the opening of calcium channels in the plasma membrane in response to depletion of a subset of calcium stores by the second messenger inositol-1,4,5-trisphosphate (IP3). Although the functional relationship between calcium store depletion and calcium entry is a well-documented phenomenon, there is little or no definitive data concerning the nature of the relevant calcium channels involved (referred to as Store Operated Channels or SOC), or the molecular mechanisms by which these channels are gated in response to calcium store depletion. Moreover, although recent data suggest that calcium entry may be regulated by pathways associated with the production of the second messengers sphingosine-1-phosphate, cyclic ADP-ribose or NAADP, the relationship between these putative pathways and calcium store depletion, and the generality of these pathways and the potential targets of these specific second messengers are either unknown or controversial. In summary, there is a significant lack of specific knowledge concerning the molecular mechanisms which regulate calcium entry into mast cells and other non-excitable cells. Because of the fundamental importance of calcium entry to mast cell function, the investigator's laboratory has embarked upon a series of experimental approaches to identify calcium entry regulatory proteins. In the preliminary data provided, experiments describe the identification and initial characterization of a novel family of putative calcium channels (CeCH proteins) which are widely expressed in non-excitable cells including mast cells. In the current application, experiments are proposed to analyze the function of these proteins in both mast cell and non-mast cell lines. In specific aim 1, studies will be performed to analyze the assembly and transport of wild type CeCH proteins, to conduct structure/function analyses to identify structural features required for proper assembly and transport, and to isolate and characterize the role of CeCH-associated proteins in CeCH function. In specific aim 2, experiments will analyze CeCH function in regulating calcium homeostasis and signaling through a combination of calcium imaging and electrophysiologic analysis of cultured cells expressing defined combinations of CeCH proteins under various types of stimulus conditions.

描述：（改编自申请人摘要）持续钙进入 肥大细胞在变态反应的启动和维持中起着关键作用， 与配体结合Fc β RI相关的反应。据信 与Fc ε RI结合相关的持续钙内流是 介导的开放的钙通道在质膜上的反应 第二信使消耗了一部分钙储存 肌醇-1，4，5-三磷酸（IP3）。虽然功能关系 钙储存耗竭和钙进入之间的关系是一个有据可查的 关于这一现象的性质，很少或没有确切的数据。 相关钙通道（称为商店经营的渠道或 SOC），或这些通道门控响应的分子机制 导致钙储存耗尽此外，虽然最近的数据表明，钙 进入可以通过与第二种蛋白质的产生相关的途径来调节。 信使鞘氨醇-1-磷酸，环ADP-核糖或NAADP， 这些推定途径与钙库耗竭之间的关系，以及 这些途径的一般性和这些特异性的潜在靶点， 第二信使要么是未知的，要么是有争议的。总之，有一个 严重缺乏有关分子机制的具体知识 其调节钙进入肥大细胞和其它非兴奋性细胞。 由于钙进入肥大细胞功能的根本重要性， 研究人员的实验室已经开始了一系列的实验， 识别钙进入调节蛋白的方法。上对初级 提供的数据，实验描述了识别和初始 一个新的推定钙通道家族（CeCH蛋白）的表征 其在包括肥大细胞在内的非兴奋性细胞中广泛表达。在 目前的应用，实验提出了分析这些功能 肥大细胞和非肥大细胞系中的蛋白质。在具体目标1中， 分析野生型CeCH的组装和转运 蛋白质，进行结构/功能分析，以确定结构 正确组装和运输所需的功能，以及隔离和 表征CeCH相关蛋白在CeCH功能中的作用。在特定 目的2，实验分析CeCH调节钙稳态的功能 以及通过钙成像和电生理学的组合来进行信号传导 分析表达确定的CeCH蛋白组合的培养细胞 在各种刺激条件下。