Cystic Fibrosis (CF) is the most common lethal genetic disease in Caucasians. It is characterized by abnormal electrolyte transport in several organs; in the lung, abnormal ion transport by airway epithelia contributes to defective pulmonary mucociliary clearance and lung disease which is the major cause of morbidity and mortality. Airway epithelia, have a reduced Cl- permeability which results from defective phosphorylation-dependent activation of apical membrane Cl-channels. The gene for the cystic fibrosis transmembrane conductance regulator (CFTR) was recently identified and shown to be mutated in patients with CF. Expression of CFTR, but not a mutant form of CFTR (delta-F508), in cultured CF airway epithelial cells corrected the Cl- channel defect. That work demonstrated a causal relationship between mutations in the CFTR gene and defective Cl- channel function, but it did not determine the function of CFTR. The goal of this application is to understand the function of CFTR. The work will test the hypothesis that CFTR is itself a cAMP-regulated Cl- channel; preliminary data are consistent with that hypothesis. However, the specific aims are designed to increase the understanding of CFTR function irrespective of whether CFTR is, or is not, a Cl- channel. CFTR will be expressed in CF cells, in nonepithelial cells, and in cells that do not normally express CFTR, in order to determine if expression of CFTR induces cAMP-activated Cl-channels. Patch clamp studies and studies of Cl-channels which are located in the apical membrane of epithelia will be used to define the properties of the cAMP-activated Cl- current and to compare it to cAMP-activated Cl- currents in cells expressing endogenous CFTR. The results should indicate whether CFTR is a channel or directly or indirectly regulates Cl- channels. They will also increase the understanding of how mutations in the CFTR gene cause the phenotypic abnormalities. Substitutions of specific amino acids will also be made to elucidate the function of CFTR. Site-directed mutations in the membrane spanning domains and adjacent sequences are capable of definitively demonstrating that CFTR is a Cl- channel and could define the amino acids that line the channel pore. Mutations in the nucleotide binding folds will provide information about the function of these interesting regions. Deletions of entire domains of the protein may give important insights into their specific functions. CFTR will also be purified and incorporated it into planar lipid bilayers. If CFTR is itself a channel, the investigators should see that incorporation of purified CFTR causes Cl- channel activity. If CFTR is not a channel, the technique will allow us to assess interactions between CFTR and Cl- channels. In either case this technique will provide one of the best ways to study CFTR.
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