The Mitochondrial ATP-Sensitive K+ Channel in Heart

描述（由申请人提供）：心脏缺血及其后遗症是导致死亡的主要原因。打开KATP通道具有强大的抗梗死作用，提供了治疗靶点的可能性和内源性心脏保护机制的见解。线粒体atp敏感的K+通道（mitoKATP）在心脏保护中起着关键作用。该项目的长期目标是揭示其在细胞信号传导中的作用及其作为保护末端执行器的作用机制。我们的方法的基本原理是基于广泛接受的结论，即涉及mitoKATP的信号通路必须发生，至少部分发生在线粒体内。具体目的是确定线粒体信号通路中激酶的拓扑结构和定位。[2]了解缺血预处理如何打开mitoKATP，以及在病理生理条件下如何调节mitoKATP的打开。确定mitoKATP打开导致信号传递到心脏保护末端效应器的机制。[4]目的确定mitoKATP打开的下游靶点和mitoKATP诱导的活性氧（ROS）信号传导。[5]确定从细胞质延伸到mitoKATP的信号序列的生化。[6]确定mitoKATP的分子特性。为了实现这些目标，实验将集中在灌注的心脏、分离的线粒体、线粒体膜的分离和膜蛋白的分离/纯化。K+输运机制将利用光散射和离子特异性荧光探针进行研究。荧光探针将研究线粒体ROS产生的生物能量学。灌注心脏治疗的效果将在分离的线粒体中使用免疫检测和K+通量测量技术进行评估。信号中间体的鉴定将利用32p标记和磷酸化的功能分析。

DESCRIPTION (provided by applicant): Cardiac ischemia and its sequelae are major causes of mortality. Opening KATP channels exerts a powerful anti-infarct effect, offering the possibilities of a therapeutic target and of insights into the mechanisms of endogenous cardioprotection. The mitochondrial ATP-sensitive K+ channel (mitoKATP) plays a pivotal role in cardioprotection. The long-term goal of this project is to uncover the mechanisms of this action with respect to its role in cell signaling and its role as an end effector of protection. The rationale for our approach is based on the widely accepted conclusion that signaling pathways involving mitoKATP must occur, at least in part, inside the mitochondrion. The specific aims are [1] To determine the topology and localization of kinases within the mitochondrial signaling pathway. [2] To determine how mitoKATP is opened by ischemic preconditioning and how mitoKATP opening is regulated under pathophysiological conditions. [3] To determine the mechanisms by which mitoKATP opening causes the signal to be transmitted to the end effectors of cardioprotection. [4] To determine the downstream targets of mitoKATP opening and mitoKATP-induced signaling by reactive oxygen species (ROS). [5] To determine the biochemistry of the signaling sequence extending from the cytosol to mitoKATP. [6] To determine the molecular identity of mitoKATP. To address these aims, experiments will focus on the perfused heart, isolated mitochondria, fractionation of mitochondrial membranes, and fractionation/purification of membrane proteins. Mechanisms of K+ transport will be studied using light scattering and ion-specific fluorescent probes. The bioenergetics of mitochondrial ROS production will be studied with fluorescent probes. Effects of treatment of the perfused heart will be evaluated in isolated mitochondria using immunodetection and K+ flux measurement techniques. Identification of signaling intermediates will utilize 32P-labeling and functional assays of phosphorylation.