The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a phosphorylation and nucleotide regulated anion channel that accounts for the cAMP stimulated Cl conductance at the apical membranes of airway epithelial cells.  We know that CF mutations eliminate, but the issue of how the loss of CFTR causes pulmonary disease poorly understood.  Significant progress has been made in defining the Cl channel properties of CFTR and the way in which cannel gating is regulated, but significant gaps remain in our understanding of CFTR function in differentiated airway epithelia.  This program aims to fill several of these gaps.  Project 1 will examine the regulation and pharmacological modulation of CFTR differentiated, polarized, Cl- secreting epithelial (including human bronchial epithelia, HBE).  Using the non-invasive techniques of current fluctuation and impedance analysis.  This project will identify the kinases and phosphatases that control apical membrane CFTR channel properties and define the way in which thee properties are altered by pharmacologic agents that open CFTR Cl channels.  Project 2 will employ biochemical and molecular methods to define the control of CFTR Cl channels by targeted kinases and phosphatases that form a regulatory complex with CFTR.  This project will examine CFTR's interaction with anchored regulators (including ezrin and EBP50) in an intracellular compartment that is enriched in CFTR and type II protein kinase A, the clathrin coted membrane vesicle.  CCVs mediate CFTR endocytosis; they re part of the regulated CFTR traffic pathway.  Project 3 seeks to provide a molecular understanding of the regulatory and structural determinants of CFTR trafficking.  It aims to identify structural information in CFTR that determines its transit between intracellular vesicles and trafficking.  Project 4 explores the interaction of CFTR with the processing and release of MUC1, the only known transmembrane mucin in human lung.  This project will define the expression, maturation and cellular distribution of MUCl in human airway epithelial and determine how the post-translational processing of MUC1 is influenced by CFTR activity and CF disease mutations.  The mechanism responsible for impaired MUC1 secretion in CF airway will be identified.  These projects interact conceptually and technically in various ways.  ll are supported by core facilities for molecular biology, cell and tissue imaging and human airway cell culture, which provide access to shared cells and regents, methods for manipulating gene expression and modern imaging techniques.  Despite the wealth of our knowledge regarding the biophysical properties and regulation of single CFTR Cl channels, there are significant gaps in our understanding of the way in which CFTR contributes to the differentiated airway cell phenotype.  This information is critical for our understanding how CFTR mutations impair the functional properties of the airway surface, and the way in which these properties can be manipulated by therapeutic intervention.