The recent identification of the cystic fibrosis (CF) gene is an important advance in our understanding of the disease. Information on the structure of the protein predicted from the amino acid sequence suggests that the CF transmembrane conductance regulator (CFTR), is a transport protein with properties similar to P-glyco proteins. Although this new information is very important, it raises several questions which must be answered to find an effective therapy for CF. Specifically, what is the function of CFTR; how does CFTR normally interact with secretory Cl- channels to allow protein kinase regulation; and how does a defect in CFTR lead to an inability of protein kinase A and C to activate secretory Cl- channels in CF tissues? The experiments proposed in this application are designed to address these questions directly. Three approaches will be applied: 1. Antibodies will be raised against specific regions of CFTR. These antibodies will be used to immunopurify the CFTR protein and to determine the specific cellular location of CFTR in airways and T84 cells. 2. A human airway cell line originating from a CF patient known to have defective Cl- channel regulation will be transfected with normal cDNA encoding CFTR to determine if normal channel regulation is restored. In addition, a rabbit thick ascending limb cell line which does not express secretory Cl- channels will also be transfected with CFTR to determine if Cl- channel function is expressed. 3. Patch clamp studies of whole cell Cl- currents and single Cl- channel activity in airways will be performed to continue studies of normal and abnormal channel regulation and development. In addition, using antibodies which bind to specific regions of CFTR the interaction between CFTR protein and Cl- secretion will be assessed.
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