Cardiac function and PIP2

磷脂酰肌醇-4，5-二磷酸（PIP 2）是肌醇-三磷酸（IP 3）的前体， (DAG)和磷脂酰肌醇-三磷酸（PIP 3）。它还锚定细胞骨架和许多信号传导 分子在质膜上，其代谢与膜运输密切相关。此外还 深刻地调节几种心脏离子转运蛋白和通道的功能。本申请 阐述了PIP 2在心脏中的调节方式以及PIP 2代谢如何与心脏中的膜周转相关。 心肌肌膜正如初步数据所建议的，我们将测试PI 4激酶是否受以下因素的调节： 丝氨酸/苏氨酸磷酸化以及脂质磷酸酶是否受表面膜插入调节 和氧依赖性蛋白水解。作为一种新的实验模型，我们已经产生了转基因小鼠， 心脏特异性2c_PI4-激酶（PI 4K 2a）过表达。这种激酶主要定位于高尔基体 和内膜，其过度表达与高度心肌肥大有关， ECC的上调。我们现在将测试心肌细胞膜的进出 影响使用（1）荧光膜染料，（2）一种新的安培法监测表面 膜融合事件，和（3）在细胞上膜片钳中的高分辨率电容测量 配置.初步数据表明，PKC可以激活内膜上的PI 4K 2Gt，从而 启动含有脂质磷酸酶的囊泡向肌膜的移动。肌膜处插入 似乎被DAG直接激活，因此随后的肌膜PIP 2的消耗将阻止 内吞作用，有利于肌膜的扩张。作为PI 4K 2（x）研究的补充，我们将测试 21型PI 4-激酶（PI 4K 213）是否是主要的肌膜PI 4-激酶，其调节是否 与心脏转运蛋白和通道的调节有关。最后，假设将被测试，PIP 2 代谢对膜张力和曲率以及对肌细胞拉伸（即， 心脏前负荷）。

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).