Immunophilins regulate SOC entry channels in pulmonary endothelial cells

描述(由申请人提供)：拟议研究的目标是确定大分子免疫亲和素FKBP51和FKBP52调节肺内皮细胞储存操作钙(SOC)进入的机制。该项目具有临床意义，因为激活特定的SOC进入通道，即ISOC通道，是导致内皮细胞间隙形成和内皮屏障通透性增加的关键步骤。通透性增加是一种血管事件，在急性和慢性炎症中都会发生，是急性肺损伤发病率和死亡率的一个促成因素。了解SOC进入的调控机制将有助于开发新的抗炎策略，但对调控知之甚少。已经观察到FKBP51和FKBP52以相反的方式调节商店操作的钙内流。FKBP51之所以引起人们的特别关注，是因为它能抑制钙库操作的钙内流，并可能导致内皮通透性降低。此外，由于FKBP51在微血管内皮细胞中优先表达，它可能是该细胞类型抵抗炎性激动剂破坏屏障的关键决定因素。最后，FKBP51是人类肺中最高水平的糖皮质激素诱导基因，其表达和活性可以通过药物调控，有可能导致有益的治疗干预措施的开发。具体地说，体外和原位实验模型将用于确定这些免疫亲和素是否是ISOC通道异源复合体的一部分(特定目标1)，特定结构域对于促进或抑制通道功能是否重要(特定目标2-3)，以及相对免疫亲和素表达是否是形成内皮细胞间隙的关键决定因素(特定目标4)。FKBP51和FKBP52的特定结构域突变体很容易获得。将使用的技术包括：差示分级、免疫沉淀和均一时间分辨荧光来确定蛋白质-蛋白质的相互作用；荧光显微镜和膜片钳电生理学来测量钙通道功能；核磁共振光谱学来测量含有脯氨酸的多肽键异构化；视频显微镜、通透性测量和阻力测量来测量体外内皮细胞间隙的形成；以及分离的灌流肺模型原位测量滤过系数(KF)、肺湿重/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氏蓝染料)。

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.