EnaC regulation in the kidney by vesicle trafficking and recycling

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Defects in ion and water regulation in the kidney have been associated with numerous diseases including, nephrogenic diabetes insipidus, hypertension, Bartter syndrome, Gitelman syndrome, pseudohypoaldosteronism type 1 and Liddle syndrome. Salt and water transport in the cortical collecting duct (CCD) of the kidney is mediated through specific channels which are tightly controlled by hormonal feedback mechanisms. The proposed studies will define and characterize novel mechanisms of channel regulation by vesicle trafficking and recycling. The studies aim to investigate whether three of the most common transporters at the CCD apical membranes, namely the epithelial sodium, potassium and water channels are co-localized and co-regulated by vesicle trafficking. Studies using a mouse CCD cell line will define the underlying physiological regulation of these trafficking events. Studies will identify specific cell machinery important in the regulated recycling of these channels. A multidisciplinary approach will first identify the subcellular vesicle trafficking compartments by immuno-cytochemical labeling and fluorescent and electron microscopic imaging. Next, the localization of channels in these vesicle compartments will be verified biochemically. The dynamics of the trafficking events will be determined using live-cell imaging and electrophysiological techniques. Finally, the mechanisms which regulate the recycling of these channels will be elucidated. By investigating the role of Rab-proteins in regulated channel recycling the essential cellular components involved in channel trafficking will be determined. These basic cell biological studies will further an understanding of water and ion channel regulation in the kidney. This will help not only to understand the essential homeostatic processes involved in salt and water balance, but more importantly, the work will provide insights into underlying defects in this regulation associated with channel misregulation which result in pathophysiological disease states.