The long-term objective of this project is to determine how the structures of sodium-dependent cotransport proteins determine their functional properties. The focus of the current proposal is to characterize at the molecular level the Na+/dicarboxylate cotransporters of the renal proximal tubule. These transporters are important to the function of the kidney in their reabsorption of Krebs cycle intermediates, and play a role in acid-base balance and organic anion excretion. The brush border Na+/dicarboxylate cotransporter has been implicated in the development of kidney stones by its regulation of urinary citrate concentrations. The principal investigator has recently cloned and sequenced a rabbit renal Na+/dicarboxylate cotransporter, NaDC-1 and the human homolog, hNaDC-1. NaDC-1 appears to correspond to the low affinity Na+/dicarboxylate cotransporter of the brush border membrane. The first Specific Aim of this study is to determine the role that histidine-106 plays in the transport of succinate by NaDC-1 and hNaDC-1. The second Specific Aim is to identify domains or residues involved in substrate selectivity and binding, by preparing chimeras between related transporters. The third Specific Aim is to clone the renal high affinity Na+/dicarboxylate cotransporter. This transporter will be used to identify residues important for high affinity substrate binding. These studies should provide fundamental information on the functional properties of this family of sodium-dependent transporters, and on the physiological role of these transporters in the kidney.
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