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 type2alpha PI4-kinase (PI4K2alpha). 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 PI4K2alpha 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 PI4K2alpha, we will test whether the type 2beta PI4-kinase (PI4K2alpha) 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).
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