Mammalian tissues, including those affected by Cystic Fibrosis, contain several mRNAs that are homologous to the segment of Band 3 mRNA encoding the membrane-spanning (anion exchange) domain. We propose to clone and sequence a number of these mRNAs that we presume encode anion transport proteins. By expression of functional mRNAs in oocytes, we will determine the type(s) of channel activity of these encoded proteins: anion exchanger, symport or channel. In parallel, we will prepare antibodies against defined segments of the derived proteins, determine by immunocytochemistry their tissue and cellular localization, and test the ability of these antibodies to block anion channels, symports, and exchangers from various tissues. We will also obtain and characterize the genomic segments encoding these presumed channel proteins. Using cloned genomic DNAs or cDNAs we will search for abnormalities in mRNA levels in tissues from Cystic Fibrosis patients. These clones will also be provided to collaborators who will search for major gene alterations and for restriction fragment length polymorphisms in genomic DNA from Cystic Fibrosis family cohorts and from normal controls. In parallel, we will continue to explore aspects of the structure of the one anion transport protein - erythrocyte Band 3 - whose sequence is known. Using in vitro mutagenesis, peptide-specific antisera and immunocytochemistry, and proteolytic digestions, we will determine the membrane topology of the protein. In vitro mutagenesis of a few selected amino acids, such as the DIDS-binding lysine, together with expression of the cloned cDNAs in oocytes or, using retroviral vectors, in 3T3 cells should define the function of several amino acids in anion exchange.
