Immunophilins regulate SOC entry channels in pulmonary endothelial cells

描述（由申请人提供）：拟议研究的目的是确定大分子量亲免素FKBP 51和FKBP 52调节肺内皮细胞中钙库操纵的钙（SOC）进入的机制。该项目具有临床意义，因为特定SOC进入通道（ISOC通道）的激活是导致内皮细胞间间隙形成和跨内皮屏障渗透性增加的关键步骤。通透性增加是一种在急性和慢性炎症中发生的血管事件，是急性肺损伤发病率和死亡率的促成因素。了解SOC进入的调节机制将有助于开发新的抗炎策略，但对调节了解甚少。已经观察到，FKBP 51和FKBP 52以相反的方式调节钙库操作的钙进入。FKBP 51是特别感兴趣的，因为它抑制钙池操作的钙进入，并可能导致内皮通透性降低。此外，由于FKBP 51优先在微血管内皮细胞中表达，因此它可能是该细胞类型对炎症激动剂造成的屏障破坏的抗性的关键决定因素。最后，FKBP 51是人肺中最高度糖皮质激素诱导的基因，并且其表达和活性可以被随机操纵，可能导致有益的治疗干预的发展。具体而言，将使用体外和原位实验模型来确定这些亲免蛋白是否是ISOC通道异源复合物的一部分（特定目的1），特定结构域是否对促进或抑制通道功能很重要（特定目的2-3），以及相对亲免蛋白表达是否是内皮细胞间间隙形成的关键决定因素（特定目的4）。FKBP 51和FKBP 52的特定结构域突变体是容易获得的。拟采用的技术包括：微分分级、免疫沉淀和均相时间分辨荧光以确定蛋白质-蛋白质相互作用;荧光显微镜和膜片钳电生理学以测量钙通道功能;核磁共振光谱法以测量含脯氨酸的肽键异构化;视频显微镜、渗透性测量和电阻测量以测量体外内皮细胞间间隙形成;离体肺灌流模型原位测定滤过系数（Kf）、肺湿/干重比、肺泡液体积分数和白蛋白通透性（支气管肺泡灌洗液中的伊文思蓝染料）。

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine the mechanisms by which the large, molecular weight immunophilins, FKBP51 and FKBP52, regulate store-operated calcium (SOC) entry in pulmonary endothelial cells. This project is of clinical significance because activation of a particular SOC entry channel, the ISOC channel, is a key step leading to the formation of inter-endothelial cell gaps and increased permeability across the endothelial barrier. Increased permeability is a vascular event that occurs in both acute and chronic inflammation and is a contributing factor to morbidity and mortality in Acute Lung Injury. Understanding regulatory mechanisms of SOC entry will aid in development of new anti-inflammatory strategies, however regulation is poorly understood. It has been observed that FKBP51 and FKBP52 regulate store-operated calcium entry in opposing manners. FKBP51 is of specific interest because it inhibits store-operated calcium entry and may lead to reduced endothelial permeability. Further, as FKBP51 is preferentially expressed in microvascular endothelial cells, it may be the key determinant of this cell type's resistance to barrier disruptin by inflammatory agonists. Finally, FKBP51 is the most highly glucocorticoid-inducible gene in human lung, and its expression and activity can be pharmacologically manipulated, potentially leading to development of beneficial therapeutic interventions. Specifically, in vitro and in situ experimental models will be used to determine whether these immunophilins are part of the ISOC channel heterocomplex (Specific Aim 1), whether specific domains are important for facilitating or inhibiting channel function (Specific Aims 2-3), and whether relative immunophilin expression is a key determinant of inter-endothelial cell gap formation (Specific Aim 4). Specific domain mutants of FKBP51 and FKBP52 are readily available. Techniques to be used include: differential fractionations, immunoprecipitation and Homogenous Time-Resolved Fluorescence to determine protein-protein interactions; fluorescence microscopy and patch-clamp electrophysiology to measure calcium channel function; nuclear magnetic resonance spectroscopy to measure proline- containing peptide bond isomerization; video microscopy, permeability measurements and resistance measurements to measure inter-endothelial cell gap formation in vitro; and the isolated, perfused lung model measuring filtration coefficient (Kf), wet/dry lung weight ratio, alveolar fluid volume fraction and albumin permeability (Evan's Blue dye in bronchoalveolar lavage fluid) in situ.