This proposal forms the center of the applicant's long-term objectives. They are to elucidate the structure and function of band 3-related proteins of the kidney, and to define their roles in normal and pathologic renal physiology at the levels of the individual cell, the local epithelium, and the entire kidney. These objectives, as met through the proposal's specific aims, will contribute to an understanding of how the renal tubular epithelial cell uses mechanisms of pHi, volume, and intracellular solute regulation present in most or all cells to modify the composition of the urine, thereby regulating pH, volume, and solute concentrations of the entire body. Towards these ends, the specific aims are to: 1. Characterize by isotopic flux studies the anion transport properties of heterologous KB3/AE1 and B3RP/AE2 expressed in Xenopus oocytes. 2. Characterize by fluorometric pHi studies the anion transport properties of heterologous KB3/AE1 and B3RP/AE2 expressed transiently in COS cells, using novel methods for supravital identification of the transfected cells. 3. Define by construction of deletions and hybrid proteins and by site-directed mutagenesis the amino acid residues of KB3/AE1 and of B3RP/AE2 responsible for ion binding and/or translocation, for pHi sensitivity of transport, and for binding of cytoskeletal proteins. 4. Develop a epithelial and nonepithelial cell lines which stably overexpress or underexpress KB3/AE1 or B3RP/AE2, or their subdomains. 5. Compare pHi regulation in the above cell lines and their parental lines. Test the hypothesis that B3RP/AE protein expression can suppress cell proliferation and/or transformation by lowering pHi. 6. Test the hypothesis that B3RP/AE protein expression is required for acute regulatory volume increase (RVI). 7. Test the hypothesis that overexpression of N-terminal cytoplasmic domains of B3RP/AE proteins will disrupt normal cytoskeletal assembly and/or polarization of plasma membrane in MDCK cells. Fulfillment of the specific aims should enhance our understanding of renal acid/base handling, concentration mechanisms, and Na+ reabsorption, and of the diverse disorders of renal cytoskeletal organization. It will provide clues to the nature of progressive renal disease, as embodied by the question of why renal epithelial cells respond to proliferative stimuli only by hypertrophy and not by hyperplasia. Finally, it will provide a avenues for rational design of new diuretic drugs, and for new cytoprotection protocols for organs at risk of ischemic damage.
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