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.
The goal of the proposed research is to determine mechanisms by which certain calcium ion channels (SOC channels) are regulated. This project is of clinical significance because SOC channels play a role in 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. Thus, understanding regulation of SOC channels may lead to the development of novel and beneficial therapeutic interventions.
