Gap Junction Trafficking to and within the Plasma Membrane

描述(申请人提供)：缺血性心脏病及其相关并发症--心脏性猝死和充血性心力衰竭--仍然是美国发病率和死亡率的主要原因。导致心律失常的缺血性疾病的病理生理学包括心肌细胞通过改变缝隙连接的定位和调节而失去电偶联。改变偶联的确切分子机制仍然难以捉摸。本研究的目的是了解缝隙连接半通道(连接蛋白)在正常和缺血条件下的细胞运动。我们的中心假设是，连接蛋白需要细胞骨架将它们定位于质膜上的特定位置，一旦进入质膜，横向扩散和其他非基于细胞骨架的通道运动的作用是有限的。这一假说将通过使用固定的和活的细胞成像技术来验证，包括高分辨率全内反射荧光(TIRF)成像，并辅以生物化学，以了解连接蛋白运输的分子机制。具体目的包括了解在氧化应激和模拟缺血条件下基于微管的连接蛋白定向靶向间盘的作用；了解肌动蛋白细胞骨架在连接蛋白靶向传递中的作用；以及定量确定连接蛋白横向扩散到质膜内其他膜区域的能力。初步数据表明，氧化应激限制了微管对皮质膜的捕获，阻止了连接蛋白向质膜的传递；肌动蛋白帮助微管定位并将离子通道定位到皮质膜的特定区域，连接蛋白的横向扩散受到高度限制。这些目的将进一步加深我们对心脏缝隙连接的调节和行为的理解。蛋白质运输的一般领域将受益于关于细胞骨架和这些通道的缺血型调节的基本新知识。此外，缝隙连接运输中的关键分子和事件将被确定为治疗靶点，以减少与缺血性心脏病相关的心律失常和功能障碍。公共卫生相关性：缺血性心脏病的临床后遗症是充血性心力衰竭和心脏性猝死，这是美国人死亡的主要原因。心力衰竭和猝死的细胞学基础都涉及心脏细胞之间的电耦合减弱。这项应用建议研究心脏细胞之间电耦合的分子机制，并确定在正常情况下和血流减少(缺血)时参与调节这种耦合的蛋白质。

DESCRIPTION (provided by applicant): Ischemic heart disease and its associated complications of sudden cardiac death and congestive heart failure remain leading causes of morbidity and mortality in the United States. The arrhythmogenic pathophysiology of ischemic disease includes loss of cardiomyocyte electrical coupling through altered localization and modulation of gap junctions. The precise molecular mechanisms underlying altered coupling remain elusive. The objective of this proposal is to understand the cellular movement of gap junction hemichannels (connexons) in normal and ischemic conditions. Our central hypothesis is that connexons require the cytoskeleton to target them to specific locations on the plasma membrane and, once in the plasma membrane, there is a limited role of lateral diffusion and other means of non- cytoskeleton based channel movement. The hypothesis will be tested by using fixed and live cell imaging techniques including high resolution total internal reflection fluorescence (TIRF) imaging, with supplementary biochemistry, to understand the molecular mechanisms of connexon trafficking. Particular aims include understanding the role of microtubule based directed targeting of connexons to intercalated discs in conditions of oxidative stress and simulated ischemia; to understand the role of the actin cytoskeleton in targeted delivery of connexons; and to determine quantitatively the capacity of connexons to diffuse laterally to other membrane regions within the plasma membrane. Preliminary data indicate that oxidative stress limits microtubule capture of cortical membrane, preventing delivery of connexons to the plasma membrane; that actin helps microtubules position and target ion channels to specific regions of the cortical membrane, and that lateral diffusion of connexons is highly restricted. These aims will further our understanding of the regulation and behavior of cardiac gap junctions. The general field of protein trafficking will benefit from fundamental new knowledge about cytoskeleton and ischemic type regulation of these channels. Furthermore, key molecules and events in the trafficking of gap junctions will be identified to be used as therapeutic targets to lessen the arrhythmias and dysfunction associated with ischemic heart disease. PUBLIC HEALTH RELEVANCE: The clinical sequelae of ischemic heart disease are congestive heart failure and sudden cardiac death which are primary causes of mortality in the United States. The cellular basis of both heart failure and sudden death involve diminished electrical coupling between heart cells. This application proposes to study the molecular mechanisms of electrical coupling between heart cells and identify proteins involved in regulating the coupling under normal conditions and during times of reduced blood flow (ischemia).