(Taken directly from the application) The long-term objective of this project is to define the molecular mechanisms that control the expression of CFTR Cl- channels at the surfaces of polarized epithelial cells. The steady-state expression of CFTR at the cell surface reflects a balance between the exocytic insertion and endocytic retrieval of CFTR as this protein continually recycles between intracellular compartments and the apical plasma membrane. The present study focuses on the functional properties of two syntaxin isoforms (i.e., syntaxins 1A and 3) and a syntaxin binding protein (i.e., an n-Sec 1 isoform) that are candidate regulators of CFTR recycling between the cell surface and intracellular compartments. Our preliminary results indicate that dominant negative mutants of syntaxins 1A and 3 inhibit apical membrane endocytosis in filter-grown colonic epithelial cells that have been permeabilized from the basolateral side with streptolysin toxin. Moreover, these mutants also markedly elevate cAMP-dependent, whole cell Cl- currents in colonic epithelial cells, as expected if they disrupt CFTR internalization from the cell surface. We propose that these syntaxin isoforms and the epithelial n-Sec 1 isoform control apical membrane recycling and thereby CFTR Cl current activity at the cell surface. We will test this hypothesis by pursuing 3 specific aims. First, we will define the roles of each syntaxin isoform in regulating apical membrane traffic, focussing specifically on their involvement in mediating CFTR internalization from the apical cell surface. Second, we will determine if one or both syntaxins regulate CFTR-dependent Cl- currents at the apical surfaces of polarized epithelial cells, as expected if they regulate CFTR internalization. As part of this aim we will also determine if, by manipulating apical membrane recycling with syntaxin mutants or antibodies, we can potentiate Cl- currents in cells that are expressing the most common CF mutation (delta F508). Third, we will define the roles of the n-Sec 1 isoform (i.e., the syntaxin binding protein) in controlling apical membrane traffic, CFTR internalization and apical Cl- currents in polarized epithelial cells. The results should provide novel insights into the molecular basis for CFTR recycling in epithelial cells; insights that may be relevant to the design of strategies for upregulating CFTR Cl- channel activity at the surfaces of CF epithelial cells.
