Molecular determinants on RyR2 involved in EC coupling

心脏的收缩是由称为EC偶联的复杂信号转导途径触发的。在这个过程中，心肌细胞的去极化激活细胞内钙释放通道;兰尼碱受体亚型2（RyR 2）从细胞内储存释放钙，释放的钙触发肌肉收缩。充分理解这一复杂的信号传导过程是心肌研究的基本目标之一。虽然RyR 2在心脏功能中的重要性已经得到了很好的证实，但由于缺乏RyR 2的转基因动物会遭受胚胎致死性，因此对参与EC偶联的RyR 2一级序列内的结构决定簇知之甚少。由该提案资助的工作将具体定义EC偶联所需的RyR 2区域。使用.利用分子生物学技术，我们将创建一组嵌合蛋白，其中RyR 2的定义片段被替换成由脑兰尼碱受体同种型组成的骨架。RyR3。虽然RyR 3能够支持钙诱导的钙释放（CICR），但它不能参与去极化诱导的EC偶联。这使它成为这些研究的理想背景。在发育不良（即RyR缺陷）肌肉细胞系中表达这些嵌合蛋白后，我们将使用高速荧光钙成像技术测试这些RyR 2-RyR 3嵌合体介导EC偶联的能力。当发现支持去极化诱导的EC偶联的嵌合体时，我们将创建含有越来越少量RyR 2的额外嵌合体，直到我们可以定义可以支持EC偶联的RyR 2序列的最小片段。在我们的发育不良骨骼肌细胞系中完成初步研究后，我们将在胚胎体中证实我们的发现，这些胚胎体表达由胚胎干细胞产生的心室蛋白，其中RyR 2的两个等位基因都被“敲除”。这将使我们能够确认我们的数据，并在心中牢固地建立其相关性。

Contraction of the heart is triggered by an intricate signal transduction pathway known as EC coupling. In this process, depolarization of the cardiac muscle cell activates an intracellular calcium release channel; the ryanodine receptor isoform 2 (RyR2) to release calcium from intracellular stores and this released calcium triggers muscle contraction. Fully understanding this elaborate signaling process is one of the fundamental goals in cardiac muscle research. While, the importance of RyR2 in heart function is well established, as transgenic animals that lack RyR2 suffer embryonic lethality, relatively little is known as to the structural determinants within the RyR2 primary sequence that are involved in EC coupling. Work funded by this proposal will specifically define the regions on RyR2 necessary for EC coupling. Using. molecular biological techniques, we will create a set of chimeric proteins where defined segments of RyR2 are substituted into a backbone made up of the brain ryanodine receptor isoform. RyR3. Although RyR3 is able to support calcium induced calcium release (CICR), it is unable to participate in depolarization induced EC coupling. This makes it the ideal background for these studies. After expressing these chimeric proteins in dyspedic (i.e. RyR-deficient) muscle cell line, we will test the ability of these RyR2-RyR3 chimeras to mediate EC coupling using high-speed fluorescence calcium imaging techniques. When chimeras that support depolarization induced EC coupling are found we will then create additional chimeras containing smaller and smaller amounts of RyR2 until we can define the smallest segment of the RyR2 sequence that can support EC coupling. After completing preliminary studies in our dyspedic skeletal muscle cell line, we will then confirm our findings in embryoid bodies which express cardiac ventricular proteins created from ES cells in which both alleles of RyR2 have been "knocked out". This will allow us to confirm our data and firmly establish its relevance in the heart.