The Mitochondrial ATP-Sensitive K+ Channel in Heart

描述(申请人提供)：心脏缺血及其后遗症是死亡的主要原因。开放KATP通道具有强大的抗心肌梗死作用，为治疗靶点和深入了解内源性心肌保护机制提供了可能性。线粒体ATP敏感性钾通道(MitoKATP)在心肌保护中起着关键作用。这个项目的长期目标是揭示这一作用的机制，包括它在细胞信号转导中的作用以及它作为保护的末端效应器的作用。我们方法的基本原理是基于一个被广泛接受的结论，即涉及mitoKATP的信号通路必须至少部分发生在线粒体内。其具体目的是[1]确定线粒体信号通路中的激酶的拓扑结构和定位。[2]确定缺血预适应如何开放mitoKATP，以及在病理生理条件下如何调节mitoKATP开放。[3]确定mitoKATP开放导致信号被传递到心脏保护的末端效应器的机制。[4]确定线粒体KATP开放的下游靶点及其通过活性氧(ROS)诱导的信号转导。[5]确定从胞浆延伸到mitoKATP的信号序列的生物化学。[6]确定mitoKATP的分子同源性。为了达到这些目标，实验将集中在心脏灌流、分离的线粒体、线粒体膜的分离以及膜蛋白的分离/纯化上。将使用光散射和离子特定的荧光探针来研究K+的转运机制。线粒体ROS产生的生物能量学将用荧光探针进行研究。使用免疫检测和K+流量测量技术，将在分离的线粒体中评估灌流心脏的治疗效果。信号中间产物的鉴定将利用~(32)P标记和磷酸化功能分析。

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.