The endothelium provides a barrier between blood constituents and the underlying tissue. This barrier plays a critically important role in regulating the transport of oxygen, nutrients and waste products between blood and tissue. Disruption of the endothelial barrier leads to an increase in permeability, a contributing factor in the development and progression of mutiple pathologies including acute respiratory distress syndrome, atherosclerosis and inflammation. Acute respiratory distress syndrome leads to impaired lung function; atherosclerosis and inflammation contribute to impaired cardiovascular function and coronary artery disease. It is estimated that cardiovascular diseases alone cause almost 40% of the deaths in North America and eventually will become the leading cause of death worldwide [N Engl J Med 2005;352:1685-95]. In order to develop preventative measures or treatment for such """"""""endothelial- dependent"""""""" diseases, it is important to first understand mechanisms underlying endothelial barrier disruption. The endothelial Isoc channel is a calcium selective ion channel whose activation is important for the formation of intercellular gaps leading to endothelial barrier disruption. While regulation of the Isoc channel requires interaction with protein 4.1, the exact nature of this interaction is poorly understood. Additionally, the molecular components which comprise the Isoc channel itself are only partly determined. The first goal of this proposal is to determine the oligomeric state and subunit stoichiometry of the endothelial Isoc channel. Here, protein cross-linking techniques will be employed to determine the oligomeric state (i.e. how many subunits), and the fluorescent technique FRET will be used to identify the subunit stoichiometry (i.e., how many of each type of subunit). These studies will be performed on cultured pumonary artery endothelial cells as well as endothelial membrane fractions obtained from the isolated perfused lung. The second goal of this proposal is to describe the nature of the interaction between the Isoc channel and protein 4.1. Here, fixed and live-cell FRET analyses will be undertaken to determine whether a direct, dynamic interaction between protein 4.1 and the Isoc channel is required for calcium permeation through the channel pore. Relevance: Endothelial cells line the inside of blood vessels forming an endothelial barrier. Endothelial barrier disruption contributes to the development and progression of multiple diseases including cardiovascular disease, a major cause of death in North America. This research proposal will study mechanisms leading to endothelial barrier disruption. ? ? ?
| Cioffi, Donna L; Lowe, Kevin; Alvarez, Diego F et al. (2009) TRPing on the lung endothelium: calcium channels that regulate barrier function. Antioxid Redox Signal 11:765-76 |
