Gap Junction Trafficking to and within the Plasma Membrane

描述（由申请人提供）：缺血性心脏病及其相关的心源性猝死和充血性心力衰竭并发症仍然是美国发病率和死亡率的主要原因。缺血性疾病的致瘤性病理生理学包括通过改变缝隙连接的定位和调节而导致的心肌细胞电耦合的丧失。改变偶联的精确分子机制仍然难以捉摸。本提案的目的是了解正常和缺血条件下间隙连接半通道（连接子）的细胞运动。我们的中心假设是，连接子需要细胞骨架将它们靶向质膜上的特定位置，并且一旦在质膜中，侧向扩散和基于非细胞骨架的通道运动的其他手段的作用有限。将通过使用固定和活细胞成像技术（包括高分辨率全内反射荧光（TIRF）成像和补充生物化学）来检验该假设，以了解连接子运输的分子机制。具体的目的包括了解微管为基础的直接靶向的连接子的氧化应激和模拟缺血条件下的闰盘的作用;了解肌动蛋白细胞骨架的作用，有针对性地提供连接子;并定量确定连接子的能力，横向扩散到质膜内的其他膜区域。初步数据表明，氧化应激限制了微管捕获皮层膜，阻止连接子传递到质膜;肌动蛋白帮助微管定位和靶向离子通道到皮层膜的特定区域，并且连接子的侧向扩散受到高度限制。这些目标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，将确定间隙连接运输中的关键分子和事件，以用作治疗靶点，以减轻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源性猝死，这是美国死亡率的主要原因。心力衰竭和猝死的细胞基础都涉及心脏细胞之间的电耦合减弱。本申请提出研究心脏细胞之间电耦合的分子机制，并鉴定在正常条件下和在血流减少（缺血）期间参与调节耦合的蛋白质。

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).