Traffic Control of Cardiac Kv Channels

 描述（由申请人提供）：我们项目的长期目标是了解（a）在基础条件下和应激反应中心肌细胞中Kv通道亚单位的运输控制，以及（B）这些运输事件如何影响Kv通道功能和心脏电活动。目前的建议是集中在慢延迟整流（IKs）通道。IKs由KCNQ 1（孔形成）和KCNE 1（调节）亚基组成。由于IKs的幅度小，激活速度慢，在基础条件下，IKs不是心肌细胞的主要复极化力。事实上，过多的IKs会增加心律失常的风险。然而，在应激条件下，当需要更多的复极化电流来缩短动作电位时程（高β-肾上腺素能张力）时，IKs振幅增加，其激活变得更快，以提供任务所需的额外复极化电流。因此，IKs是一种“复极储备”：它有助于心脏科普压力。这一建议是基于我们最近在成人心室肌细胞中的发现：在基础条件下，KCNQ 1和KCNE 1在很大程度上相互分离。KCNE 1位于细胞表面，而KCNQ 1位于细胞质条纹区室。未发表的实验进一步表明，KCNQ 1和KCNE 1在不同的粗糙ER域中翻译，并通过不同的路线到达各自的目的地。为了探索控制KCNQ 1和KCNE 1在成人心室肌细胞中分布模式的途径和靶向/锚定机制，我们提出了3个具体目标：（1）描述KCNQ 1和KCNE 1在其生物发生过程中穿越的亚细胞区室之间的时空关系。（2）确定KCNQ 1在成年心室肌细胞中的条纹区室的性质。（3）确定生理条件下成年心室肌细胞如何形成功能性IKs通道。为了实现这些目标，我们将在成年豚鼠心室肌细胞中表达荧光标记的KCNQ 1和KCNE 1，并使用“光学脉冲追踪”策略来跟踪它们在不同条件下通过亚细胞区室的运动。这些共聚焦实验由膜片钳记录（以测定IKs通道功能）和蛋白质生物化学（以定量不同亚细胞区室中的蛋白质水平）补充。我们认为IKs通道在膜通道中并不独特：其他多组分通道可能在生物发生的后期阶段单独翻译和组装其组分，并且已经显示出几种通道具有细胞内储库，可以在需要时快速将新通道递送到细胞表面。这里获得的知识将为重新思考膜通道亚基如何在心脏和其他细胞类型中翻译，储存和组装铺平道路。

 DESCRIPTION (provided by applicant): The long-term objectives of our project are to understand (a) the traffic control of Kv channel subunits in cardiac myocytes under basal conditions and in response to stresses, and (b) how these trafficking events impact on Kv channel function and cardiac electrical activity. The current proposal is focused on the slow delayed rectifier (IKs) channel. IKs is composed of KCNQ1 (pore-forming) and KCNE1 (regulatory) subunits. Due to its small amplitude and slow rate of activation, IKs is not a major repolarizing force in cardiac myocyte under basal conditions. In fact, too much IKs can increase the risk for arrhythmia. However, under stressful conditions when more repolarizing current is needed to shorten the action potential duration (high β-adrenergic tone), the IKs amplitude is increased and its activation becomes faster to provide extra repolarizing current needed for the task. Therefore, IKs is a 'repolarization reserve': it helps the heart cope with stresses. This proposal is based on our recent findings in adult ventricular myocytes: under basal conditions KCNQ1 and KCNE1 are largely segregated from each other. KCNE1 is on the cell surface, while KCNQ1 is in a cytosolic striation compartment. Unpublished experiments further suggest that KCNQ1 and KCNE1 are translated in different rough ER domains, and travel by different routes to their respective destinations. To explore the pathways and targeting/anchoring mechanisms that control the distribution patterns of KCNQ1 and KCNE1 in adult ventricular myocytes, we propose 3 Specific Aims: (1) To delineate the spatiotemporal relationships among the subcellular compartments traversed by KCNQ1 and KCNE1 during their biogenesis, (2) To determine the nature of KCNQ1 striation compartment in adult ventricular myocytes, (3) To determine how functional IKs channels are formed in adult ventricular myocytes under physiological conditions. To accomplish these Aims, we will express fluorescently tagged KCNQ1 and KCNE1 in adult guinea pig ventricular myocytes, and use an 'optical pulse-chase' strategy to follow their movements through subcellular compartments under varying conditions. These confocal experiments are supplemented by patch clamp recording (to assay IKs channel function), and protein biochemistry (to quantify protein levels in different subcellular compartments). We believe IKs channel is not unique among membrane channels: other multiple-component channels may have their components translated separately and assembled at a late stage of biogenesis, and several channels have been shown to have intracellular reservoirs that can quickly deliver new channels to the cell surface in times of need. Knowledge gained here will pave the way for rethinking how membrane channel subunits are translated, stored, and assembled in cardiac and other cell types.