Disorders of parathyroid hormone which result in significant morbidity and mortality are a common feature of chronic and end-stage renal disease. Parathyroid hormone (PTH), a key regulator of calcium and phosphorous homeostasis, controls the levels of sodium-phosphate cotransporters NaPi2a and NaPi2c and the calcium channel TRPV5 in the apical menbrane of renal tubular cells. The trafficking of these cotransporters/channels to and from the apical cell membrane represents the final common pathway through which PTH mediates its physiological action. The precise molecular mechanisms whereby PTH regulates apical trafficking, however, remain unknown. The goal of this project is to determine ho PTH regulates trafficking of NaPi transporters and the TRPV5 channel at the apical membrane of renal tubular cells. Preliminary data obtained thus far indicate that there is differential trafficking of NaPi2a, NaPi2c and TRPV5 in response to PTH. My hypothesis is that these transporters and channels interact differently with the cellular components crucial for trafficking: scaffolding (PDZ) proteins, the actin cytoskeletonand motor proteins. Trafficking will be studied using a wide variety of techniques. Dynamic regulation of trafficking in living cells will be examined using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching. These imaging techniques will be complemented by investigation of the effects of PTH on the cellular localization of these transporters/channels in native tissue. Interactions of the transporters with PDZ proteins will be studied using co-immunoprecipitation. The role of the actin cytoskeleton will be dissected using agents that modify the cytoskeleton. The functional effects of PTH on TRPV5 channel s will be investigated using electrophysiology. This project will allow me to couple the elctrophysiology skills I acquired as a graduate student with the high-resolution, dynamic imaging methods I have learned in my sponsor's laboratory. In addition, I will learn new biochemical techniques and advanced biophysical methods. Ultimately, I intend to acquire the skills necessary to establish myself as an independent investigator.
This work will provide insights into PTH regulation of calcium and phosphorous homeostasis which is critical for normal cellular function. This knowledge may ultimately lead to the development of novel therapies to treat altered calcium and phosphorous metabolism.
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