In order to maintain a constant body pH, the kidney transports H+ and HCO3 in a highly regulated manner. My previous research examined the cellular regulation of proton secretion in the turtle urinary bladder, a model for the mammalian distal nephron. The cells that are responsible for proton secretion in this part of the nephron are the intercalated cells, which comprise about 25% of this epithelium. These cells have an apical proton-translocating ATPase which transports H+ into the tubular lumen and a basolateral Cl/HCO3 exchanger which restores HCO3- to the blood. The distal tubule is also capable of secreting HCO3-. The HCO3- secreting cells are a different morphologic and functional type of intercalated cell, with a basolateral proton pump and an apical Cl/HCO3 exchanger. Comparison of the apical and basolateral Cl/HCO3 exchangers of intercalated cells is likely to yield interesting information about the signals governing the sorting of these proteins to their respective apical and basolateral membrane domains. The basolateral exchanger has been identified with antibodies to band 3, the Cl/HCO3 exchanger of erythrocytes. The goal of my current research is to identify and characterize the apical Cl/HCO3 exchanger of the HCO3- secreting intercalated cells. The first goal of this research is to purify the apical Cl/HCO3 exchanger in order to obtain N terminal sequence and to make specific antibodies. In order to purify this protein, I must first purify membranes containing this protein because of the presence of other Cl/HCO3 exchangers in the kidney. This part of the project is nearly completed. The second step in the purification of any protein is the development of a functional assay for the protein. I have developed a functional assay for the apical Cl/HCO3 exchanger which I will use in purification. The ultimate goal of this project is to examine the signals involved in sorting the apical Cl/HCO3 exchanger to its proper target, the apical cell membrane. In particular, I propose to examine the differences in cytoskeletal interaction between the apical and basolateral Cl/HCO3 exchangers. These experiments require a cell culture system for doing pulse chase and other classical experiments of cell biology. This part of the project has already been completed. They also require a specific antibody to both the apical and basolateral exchangers. I will purify the apical exchanger to develop specific antibodies.
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