Molecular Determinants of Lung Endothelial Isoc Activity

项目概述：内皮提供了血液成分和底层组织之间的屏障。这一屏障在调节氧气、营养物质和废物在血液和组织之间的运输方面起着至关重要的作用。内皮屏障的破坏导致通透性增加，这是多种病理的发生和发展的一个因素，包括急性呼吸窘迫综合征、动脉粥样硬化和炎症。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有助于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地区近40%的死亡，并最终将成为世界范围内的主要死亡原因[N Engl J Med 2005； 32:1685-95]。为了制定预防措施或治疗这种“内皮依赖性”疾病，首先了解内皮屏障破坏的机制是很重要的。内皮Isoc通道是一种钙选择性离子通道，其激活对于形成细胞间隙导致内皮屏障破坏至关重要。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尚不清楚。此外，构成Isoc通道本身的分子组分仅部分确定。本提案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。在这里，蛋白质交联技术将被用来确定寡聚状态（即有多少亚基），荧光技术FRET将被用来确定亚基化学计量（即，每种亚基有多少）。这些研究将在培养的肺动脉内皮细胞以及从离体灌注肺中获得的内皮膜部分上进行。本提案的第二个目标是描述Isoc通道和蛋白4.1之间相互作用的性质。在这里，将进行固定细胞和活细胞FRET分析，以确定钙通过通道孔渗透是否需要蛋白4.1和Isoc通道之间的直接动态相互作用。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生和进展，包括心血管疾病，这是北美死亡的主要原因。本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.