The Mitochondrial ATP-Sensitive K+ Channel in Heart

描述（由申请方提供）：心肌缺血及其后遗症是死亡的主要原因。打开KATP通道可发挥强大的抗梗死作用，为治疗靶点和深入了解内源性心脏保护机制提供了可能性。线粒体ATP敏感性钾通道（mitoKATP）在心脏保护中起着关键作用。该项目的长期目标是揭示这种作用的机制，即其在细胞信号传导中的作用及其作为保护的末端效应物的作用。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，至少部分地，在线粒体内。具体目标是[1]确定线粒体信号通路中激酶的拓扑结构和定位。[2]确定缺血预处理如何开放mitoKATP以及在病理生理条件下mitoKATP开放如何调节。[3]确定mitoKATP开放导致信号传递到心脏保护末端效应器的机制。[4]确定mitoKATP开放和mitoKATP诱导的活性氧（ROS）信号传导的下游靶点。[5]确定从细胞质延伸到mitoKATP的信号序列的生物化学。[6]确定mitoKATP的分子特性。为了解决这些目标，实验将集中在灌注心脏，分离的线粒体，线粒体膜的分馏，和膜蛋白的分馏/纯化。K+运输机制将使用光散射和离子特异性荧光探针进行研究。线粒体ROS产生的生物能量学将用荧光探针进行研究。将使用免疫检测和K+通量测量技术在分离的线粒体中评价灌注心脏的治疗效果。信号传导中间体的鉴定将利用32 P标记和磷酸化的功能测定。

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.