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).

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL067942-05
Application #
7150002
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Lathrop, David A
Project Start
2003-01-15
Project End
2008-11-30
Budget Start
2006-12-01
Budget End
2008-11-30
Support Year
5
Fiscal Year
2007
Total Cost
$369,790
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Physiology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Hilgemann, Donald W (2014) Fishing for holes in transporters: how protons breach the Na/K pump security gates. J Gen Physiol 143:437-41
Hilgemann, Donald W (2014) Cardiac electrophysiology delivered a ""grand slam"" by angiotensin II: the third explanation of transmural cardiac electrical activity gradients. Biophys J 106:2288-90
Lin, Mei-Jung; Fine, Michael; Lu, Jui-Yun et al. (2013) Massive palmitoylation-dependent endocytosis during reoxygenation of anoxic cardiac muscle. Elife 2:e01295
Fine, Michael; Lu, Fang-Min; Lin, Mei-Jung et al. (2013) Human-induced pluripotent stem cell-derived cardiomyocytes for studies of cardiac ion transporters. Am J Physiol Cell Physiol 305:C481-91
Hilgemann, Donald W; Fine, Michael; Linder, Maurine E et al. (2013) Massive endocytosis triggered by surface membrane palmitoylation under mitochondrial control in BHK fibroblasts. Elife 2:e01293
Lariccia, Vincenzo; Fine, Michael; Magi, Simona et al. (2011) Massive calcium-activated endocytosis without involvement of classical endocytic proteins. J Gen Physiol 137:111-32
Hilgemann, Donald W; Fine, Michael (2011) Mechanistic analysis of massive endocytosis in relation to functionally defined surface membrane domains. J Gen Physiol 137:155-72
Fine, Michael; Llaguno, Marc C; Lariccia, Vincenzo et al. (2011) Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains. J Gen Physiol 137:137-54
Hilgemann, Donald W (2007) Local PIP(2) signals: when, where, and how? Pflugers Arch 455:55-67
Yaradanakul, Alp; Hilgemann, Donald W (2007) Unrestricted diffusion of exogenous and endogenous PIP(2 )in baby hamster kidney and Chinese hamster ovary cell plasmalemma. J Membr Biol 220:53-67

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