Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Patch clamp and reconstitution studies have demonstrated that this gene encodes a protein kinase and nucleotide regulated Cl selective channel. The role of the CFTR in transepithelial sodium chloride absorption and secretion explains the CF phenotype: defective regulation of apical Cl conductance by cAMP signaling pathways. Yet, the manner in which this Cl channel defect causes airway disease is not yet clear. At the apical membranes of airway epithelia, the regulation of Cl conductance results from two phenomena: the trafficking and insertion of CFTR Cl channels into the apical membrane, and regulation of CFTR in the membrane. Both acute regulation of plasma membrane CFTR channel activity and membrane trafficking of vesicles containing CFTR have been demonstrated in different CFTR expression systems. Kinase regulation of both processes is impaired in cells expressing mutant CFTRs. The overall aim of our studies to identify the structural and regulatory features of CFTR that determine its insertion and retrieval at the plasma membrane, and the way in which CFTR traffic influences the activity of other ion channels. The primary hypothesis underlying this proposal is that CFTR contains regulatory and structural information that determines its trafficking. A close correlation exists between CFTR insertion and retrieval an the regulation of CFTR Cl channel gating. Despite the wealth of our knowledge regarding the structural features of CFTR that determine channel gating, we know little of the molecular determinants of its trafficking and the way in which these events influence its interaction with other channels or cellular processes. Accordingly, we will use CFTR-expressing cells, human airway cell lines and primary airway cell cultures to identify the regulatory and structural features of CFTR that determine it sinser6tion and retrieval at the plasma membrane. The studies will be carried out using simultaneous measurements of Cl current and membrane capacitance, to assess the effects of phosphorylation and traffic regulatory proteins on these CFTR trafficking events. This knowledge is necessary for understand how the apical membrane content of wild type and mutant CFTRs is determined, for the understanding and treatment of CF lung disease, and to potentially modulate the plasma membrane content of CFTR subsequent to gene transfer.
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