Polarized trafficking, appropriate surface expression and disparate regulation of at least two different potassium channels on opposite membrane domains of the renal cortical collecting duct (CCD) principal cell insure an efficient potassium secretion process and potassium homeostasis. Here, we propose to elucidate the molecular mechanisms governing polarized targeting and surface expression of the basolateral CCD channel, Kir 2.3. Our previous work suggests a hierarchical trafficking program, involving a novel biosynthetic sorting process and dynamic, PDZ- dependent retention at the basolateral membrane. To critically test this hypothesis, a stepwise multidisciplinary approach, combining molecular genetics, cellular biology, electrophysiology and transgenics, will be employed to answer the following questions: 1. How is the basolateral trafficking signal in Kit 2.3 interpreted within the biosynthetic sorting pathway? This aim is designed to critically test the role of novel biosynthetic sorting machinery candidates. 2. Does internalization of Kit 2.3 occur via clathrin-dependent mechanism, involving a direct interaction with the # subnnit of AP2 adaptor complex.
This aim i s designed to elucidate the molecular mechanisms involved in endocytotic trafficking of Kit 2.3, providing a context to understand how PDZ interactions regulate Kit 2.3 expression. 3. Does interaction with the Lin-7/CASK PDZ complex coordinate basolateral expression of Kit 2.3 by limiting endosomal trafficking. In this aim, plasma membrane turnover rate and intracellular trafficking of externally tagged channels will be assessed in the absence and presence of dominant interfering Lin-7 constructs. 4. How is interaction with MOPP, a unique PDZ protein, regulated to control surface expression of Kir 2.3? This aim is designed to test the hypothesis that MOPP acts as a natural negative regulator of Lin 7 PDZ scaffolding- complexes. 5. Does Lin-7 interaction regulate Kir 2.3 expression in the CCD during potassium adaptation? In this aim, we will determine if Lin -7 interaction underpins the physiological regulation of Kir 2.3. Wild-type and Lin-7 knockout mice will be studied. These studies represent a timely and important extension of the principal investigator's work, and should ultimately provide considerable insight into the basis of renal K handling and K homeostasis in health and disease while illuminating new and presently unexplored mechanisms controlling membrane-protein sorting in the kidney.
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