Role of Myosin VI in CFTR Endocytosis in the Intestine

Children'S Hosp Pittsburgh/Upmc Hlth Sys, Pittsburgh PA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The long term goal of the PI is to fill critical gaps in our understanding of the physiologic relevance of trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to anion transport in the intestine and its relevance to the pathogenesis of secretory diarrhea. CFTR is central to the pathogenesis of secretory diarrhea and cystic fibrosis. Though cAMP and cGMP-dependent phosphorylation regulate CFTR and intestinal anion secretion, in studies supported by this K08 award, we provided the first evidence that cAMP and cGMP also regulate surface pools of CFTR and anion secretion in the native small intestine by insertion and trafficking of endosomal CFTR into the plasma membrane of enterocytes. While up-regulation of CFTR function on the surface of enterocytes is accepted to underly the pathogenesis of secretory diarrhea, the role that trafficking plays in regulating surface pools of functional CFTR channels and anion secretion is not appreciated. The studies outlined over the last two years are a natural extension of work proposed in the K08 application to examine the role that actin-dependent endocytosis plays in regulating surface pools of CFTR in the small intestine following cyclic nucleotide activation. Instead of pharmacologic interventions to examine actin-dependent endocytosis of CFTR in the intestine, we will employ superior approaches and two independent but relevant physiologic model systems to examine the role of an actin-binding motor protein in regulating CFTR endocytosis in the intestine. Myosin VI, the only inwardly directed actin-binding motor protein, is enriched on the brush border of enterocytes, in clathrin coated pits and vesicles, regulates clathrin dependent endocytosis and is a prime candidate to regulate CFTR endocytosis. We will employ a mouse model of targeted disruption of Myosin VI (Snell's waltzer) to examine surface pools of CFTR and fluid transport following activation with the cGMP agonist Heat Stable Enterotoxin (STa). We will also develop a polarized intestinal cell model previously characterized for the study of intestinal myosins (CaCo-2BBe) and use this model to examine the role of Myosin VI in regulating the endocytosis of CFTR from its natural residence on the apical membrane. This model will also be characterized for our long term studies of CFTR traffic and anion transport. We will employ cell biological approaches including surface biotinylation, Western Blotting, immunofluorescece and immunoelectron microscopic localization in all animal and cell culture studies in conjunction with electrophysiologic approaches to understand the physiologic role of Myosin VI in regulating CFTR endocytosis and.anion secretion in the intestine. The additional funds provided by this award will enable the PI to expand investigations and develop collaborations as she pursues these revised specific aims. [unreadable] [unreadable]