In cystic fibrosis (CF), mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are associated with defective cAMP and acid-stimulated duodenal bicarbonate secretion, and defective cAMP and cGMP-activated fluid secretion in the intestine. On the other hand, increased activation of CFTR by cAMP and cGMP agonists elicit massive fluid secretion that result in secretory diarrhea. But the mechanisms responsible for increased or decreased activation of CFTR on the apical plasma membrane under physiological conditions in the intestine are unknown. Protein kinase A and G (PKA, PKG) phosphorylation is accepted as central to CFTR regulation. But this dogma assumes that CFTR is confined to the apical plasma membrane. We hypothesize that under physiological conditions in the intetsine, cell and region specific trafficking regulate the number and function of CFTR channels on the cell surface and contribute to disease pathogenesis. This hypothesis is supported by the following observations: (1) CFTR expression is cell and region-specific in the intestine (2) more than 50% of CFTR is in intracellular organelles in native enterocytes and supports constituitive and regulated trafficking in vivo (3) PKA and PKG regulate CFTR trafficking to the cell surface and fluid secretion in a cell and region specific manner in the intestine (4) in the duodenum, cAMP and luminal acid regulate CFTR trafficking and bicarbonate secretion (5) the endocytosis of CFTR from the apical plasma membrane in the intestine of transgenic mice lacking expression of the actin-binding motor myosin VI (Myo6 (sv/sv))is markedly defective, leads to its accumulation on the cell surface and increased CFTR-activated fluid secretion elicited by the cGMP- agonist Heat Stable Enterotoxin in vivo. Over the next five years, we propose to expand these observations and further elucidate the biological relevance of region and cell-specificity of CFTR trafficking, the role of luminal acid in regulating CFTR trafficking and function in the duodenum and the mechanism by which myosin VI regulates CFTR trafficking. We plan an integrative approach that incorporates state of the art cell biological structural, biochemical, and immunological studies including RNA silencing and in vivo perfusion and electrophysiology. These approaches, in conjunction with in vivo models of rat, and transgenic Myo6 (sv/sv) mouse intestine, polarized secretory and absorptive intestinal cells expressing endogenous CFTR and WT (wild type), DF508 and N287Y mutant CFTR-expressing cells will facilitate a comprehensive investigation to meet the defined goals of this proposal. PUBLIC HEALTH RELEVANCE In cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator CFTR chloride channel fails to reach the plasma membrane of cells and in secretory diarrhea, CFTR function on the membrane of intestinal cells is increased. But the mechanisms that lead to CF or secretory diarrhea are unknown. This proposal seeks to understand the mechanisms that regulate CFTR movement into to the membrane under physiological conditions in the intestine in order to elucidate how dysfunction of CFTR leads to either CF or secretory diarrhea.