Cystic fibrosis is a disease involving certain epithelial cells' inability to maintain hydrated secretions at a viscosity that can be easily passed through luminal spaces. It has recently been demonstrated that epithelial cells from CF individuals have a defective chloride transport system that affects the viscosity of their secretory products. These newer findings have provided a clear indication that the possible gene defect in cystic fibrosis involves the chloride channel transport system. With this in mind, we have devised a strategy for cloning the chloride channel protein and propose strategies for isolating other members of the complex. This project has three primary goals. First, we will identify, isolate, and characterize a full-length cDNA encoding for the chloride channel. Included in this objective are the determinations of the cDNA's nucleotide sequence and the deduced amino acid sequence of the Cl- channel protein. Information obtained from an analysis of the amino acid sequence will provide important data on the structure of the protein itself. In addition, the cDNA will be used as a probe to identify the chromosomal location of the chloride channel in humans to determine if it maps to chromosome 7, the location of the principal defect in CF. The basic strategy for accomplishing this objective involves constructing a cDNA library from poly A+ RNA employing techniques screened initially with a specific oligonucleotide that is the antisense sequence to the mRNA that codes for the DIDS binding peptide used to make the antibody described in Project 1. A second strategy for screening is to use this antibody and/or any additional polyclonal antibodies to the Cl- channel developed in Project 1. The second major experimental goal of the project will be to insert the full-length cDNA clone into (i) suitable prokaryotic expression vectors for characterizations and (ii) vectors that can be used to transfect eukaryotic cell in order to get both transient and stable expression of the chloride channel for additional functional studies. The third specific aim is to utilize the rC1 channel itself as a possible ligand with which to identify putative regulatory proteins that govern the functional activity of the channel.