MOLECULAR BIOLOGY OF CYSTIC FIBROSIS

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We will utilize the strengths of our multidisciplinary CF Research Group to study the processing, trafficking and function of CFTR, the role of individual domains, the interaction with other ion channels, and other Cl- channel pathways. Project I: Investigation of Alternate Cll Channels. The goal is to determine whether the human ClC-2 Cl- channel can ameliorate the Cl- defect in CF. Specific aims will: 1. complete the cloning of the human form, 2. characterize ClC-2 generated Cl- currents, 3. determine the cellular location of ClC-2, and 4. determine the role of ClC-2 in airway cells. Project II: Na+ and Cation Channels in CF. Na+ reabsorption is abnormally high in patients with CF. The goals are to 1. to evaluate the interaction between cation channels and CFTR, 2. to evaluate whether nucleotide-gated cation channels contribute to Na+ reabsorption in CF, and 3. to clone, then characterize the pharmacologic and electrophysiologic properties of intestinal nucleotide-gated cation channels. Project III: Interaction of CFTR with ORCC. CFTR regulates outwardly rectifying Cl- channels (ORCCs). The following questions will be addressed: 1. What is the mechanism of coupling between ORCC and CFTR? 2. What structural domains of CFTR are critical for the regulatory interaction between CFTR and ORCCs. 3. Which amino acids define the pore of CFTR. Project IV: Functional Analysis of CFTR. A key unknown in CF is which amino acids are lining the CFTR Cl- channel pore. Two goals will be achieved: 1. identify amino acids important to Cl channel activity and 2. to identify residues that may interact as salt bridges in CFTR. Project V: Folding and Trafficking of STE6 and CFTR in Yeast. CFTR and the Saccharomyces cerevisiae transporter STE6 are members of a superfamily of ATP binding cassette proteins. Major goals include: 1. the isolation of misfolding mutants of STE6, 2. identification of the mechanism of membrane insertion, folding, quality control, trafficking, and degradation of ABC proteins, 3. manufacture of STE6-CFTR chimeras to study folding and quality control machinery, 4. high level expression of CFTR, and 5. probing the intramolecular architecture of STE6. Project VI: Production and Vectorial Characterization of Native CFTR. One of the problems with hamper functional studies is a lack of sufficient amounts of CFTR. The goals are to: 1; coexpress CFTR and molecular chaperones in baculoviral/insect systems, 2. use rapidly proliferating parasitic organisms as eukaryotic overexpression systems, 3. utilize E. coli to produce large quantities of CFTR, 4. incorporate purified CFTR into liposomal vesicles and 5. determine the extent to which phosphorylation, ATP hydrolysis and counterion flow are required for CFTR function. There will be two cores: Molecular Biology and Administrative.