Cardiac function and PIP2

超出提供的空间。磷脂酰肌醇-4，5-二磷酸(PIP2)是三磷酸肌醇(IP3)、甘油二酯(DAG)和三磷酸磷脂(PIP3)的前体。它还将细胞骨架和众多信号分子锚定在质膜上，其代谢与膜运输密切相关。此外，它还深刻地调节了几种心脏离子转运体和通道的功能。这项应用解决了PIP2在心脏中是如何调节的，以及PIP2的代谢如何与心肌肌膜的膜周转有关。根据初步数据，我们将测试PI4-激酶是否受丝氨酸/苏氨酸磷酸化的调节，以及脂磷酸酶是否受表膜插入和氧依赖蛋白分解的调节。作为一种新的实验模型，我们建立了心脏特异性PI4K2a高表达的转基因小鼠。该激酶主要定位于高尔基体和内膜，其过度表达与高级别心肌肥厚和ECC上调有关。我们现在将使用(1)荧光膜染料，(2)监测表面膜融合事件的新的安培方法，以及(3)在细胞膜片钳配置中的高分辨率电容测量，来测试进出心肌肌膜的膜如何受到影响。初步数据表明，PKC可能激活内膜上的PI4K2Gt，从而启动含有脂磷酸酶的囊泡向肌膜的移动。在肌膜上的插入似乎是由DAG直接激活的，因此随后肌膜PIP2的耗尽将阻止内吞作用并有利于肌膜的扩张。作为对PI4K2(X)研究的补充，我们将测试21_PI4-激酶(PI4K213)是否是主要的肌膜P4-激酶，以及它的调节是否与心脏转运体和通道的调节有关。最后，PIP2代谢对膜张力和曲率以及心肌细胞拉伸(即心脏前负荷)的内在敏感性的假设将得到检验。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. Phosphatidylinositol-4,5,-bis-phosphate (PIP2) is the precursor of inositol-trisphosphate (IP3), diacylglycerol (DAG), and phosphatidylinositol-trisphosphate (PIP3). It also anchors cytoskeleton and numerous signaling molecules at the plasmalemma, and its metabolism is closely coupled to membrane trafficking. In addition, it modulates profoundly the function of several cardiac ion transporters and channels. This application addresses how PIP2 is regulated in heart and how PIP2 metabolism is related to membrane turnover at the cardiac sarcolemma. As suggested by Preliminary Data, we will test whether PI4-kinases are regulated by serine/threonine phosphorylation and whether lipid phosphatases are regulated by surface membrane insertion and oxygen-dependent proteolysis. As a new experimental model, we have generated transgenic mice with cardiac-specific over-expression of the type2c_ PI4-kinase (PI4K2a). This kinase localizes primarily to Golgi and internal membranes, and its over-expression is associated with high-grade cardiac hypertrophy and up-regulation of ECC. We will now test how membrane trafficking to and away from the cardiac sarcolemma is affected using (1) fluorescent membrane dyes, (2) a new amperometric method to monitor surface membrane fusion events, and (3) high resolution capacitance measurements in on-cell patch clamp configuration. Preliminary Data suggests that PKC's may activate PI4K2Gt on internal membranes, thereby initiating movement of vesicles containing lipid phosphatases to the sarcolemma. Insertion at the sarcolemma appears to be activated directly by DAG, whereby subsequent depletion of sarcolemmal PIP2 would prevent endocytosis and favor the expansion of the sarcolemma. Complementary to studies of PI4K2(x, we will test whether the type 21_ PI4-kinase (PI4K213) is the major sarcolemmal PI4-kinase and whether its regulation may be tied to the regulation of cardiac transporters and channels. Finally, the hypothesis will be tested that PIP2 metabolism is inherently sensitive to membrane tension and curvature, as well as to myocyte stretch (i.e. the cardiac preload). PERFORMANCE SITE ========================================Section End===========================================