Molecular Determinants of Lung Endothelial Isoc Activity

描述（由申请人提供）：项目概述：内皮在血液成分和底层组织之间提供屏障。这种屏障在调节血液和组织之间的氧气、营养物质和废物的运输方面起着至关重要的作用。内皮屏障的破坏导致渗透性增加，这是包括急性呼吸窘迫综合征、动脉粥样硬化和炎症在内的多种病理学的发展和进展的一个促成因素。急性呼吸窘迫综合征导致肺功能受损;动脉粥样硬化和炎症导致心血管功能受损和冠状动脉疾病。据估计，仅心血管疾病就造成了北美近40%的死亡，并最终将成为全世界死亡的主要原因[N Engl J Med 2005;352：1685-95]。为了开发针对此类“内皮依赖性”疾病的预防措施或治疗，重要的是首先理解内皮屏障破坏的潜在机制。内皮Isoc通道是一种钙选择性离子通道，其激活对于导致内皮屏障破坏的细胞间间隙的形成是重要的。虽然Isoc通道的调节需要与蛋白4.1相互作用，但这种相互作用的确切性质知之甚少。此外，组成Isoc通道本身的分子组分仅部分确定。该建议的第一个目标是确定内皮Isoc通道的寡聚状态和亚基化学计量。在此，将采用蛋白质交联技术来确定寡聚状态（即有多少亚基），并且将使用荧光技术FRET来鉴定亚基化学计量（即，每种类型的亚单位有多少）。这些研究将在培养的肺动脉内皮细胞以及从分离的灌注肺获得的内皮膜组分上进行。该提案的第二个目标是描述Isoc通道与蛋白质4.1之间相互作用的性质。在这里，固定和活细胞FRET分析将进行，以确定是否蛋白质4.1和Isoc通道之间的直接，动态的相互作用是钙渗透通过通道孔所必需的。相关性：内皮细胞排列在血管内部，形成内皮屏障。内皮屏障破坏有助于多种疾病的发展和进展，包括心血管疾病，这是北美的主要死亡原因。这项研究计划将研究导致内皮屏障破坏的机制。

DESCRIPTION (provided by applicant): Project Summary: 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.