A major task of the kidney tubule is to reabsorb filtered HCO-3 and generate """"""""new"""""""" HCO-3, thereby preventing a fatal metabolic acidosis. About 80 percent of HCO-3 reabsorption and generation occurs in the proximal tubule (PT), which secretes H+ into the tubule lumen, titrating HCO-3 to CO2 + H2O. After entering the PT cell, the CO2 + H2O regenerate HCO-3, which exits across the basolateral membrane via the electrogenic Na/HCO3 cotransporter (NBCe1-A). Since the Pl's laboratory reported the expression cloning of this cotransporter nearly 5 yrs ago, both """"""""pancreatic"""""""" (NBCe1-B) and """"""""brain"""""""" (NBCe1-C) splice variants have been identified. These cotransporters play key roles in HCO-3 transport by other epithelia, and in pHi regulation by many cell types. NBCe1 is part of the Bicarbonate Transporter (BT) superfamily, along with the C1-HCO3 exchangers (AEs), two other Na+ -coupled HCO-3 transporters (the electroneutral NBC and the Na+ -driven C1-HCO3 exchanger), related proteins not yet fully characterized, and at least one new gene (known from human genome sequence). The major goal of this project is to elucidate the molecular physiology of electrogenic NBCs, particularly in the kidney. An ancillary goal is to elucidate the expression of other Na+ -coupled HCO-3 transporters in key renal cell types. The proposed work has three aims: (i) Develop molecular tools. We will obtain the cDNA for a new NBCe-related sequence identified in the genome, extend our panel of type-specific antibodies, and determine the localization of Na+ -coupled HCO-3 transporters in the kidney. (ii) Determine properties of wild- type electrogenic NBCs. Using heterologous expression in oocytes, we will determine the function of two cDNA clones likely to encode electrogenic NBCs. We will also determine the stoichiometry of the electro-genic NBCs; assess their dependence on Na+, HCO-3 and pH; ask whether they transport CO=3; characterize the interaction between NBCe1 and carbonic anhydrase II; and examine the action of PKA on NBCe1 in oocytes. (iii) Analyze structure-function relationships. We will determine the structural requirements for extra- and intracellular DIDS sensitivity, ask whether conserved putative DIDS-reaction motifs are involved in electrostatic trapping of HCO-3, assess naturally occurring human NBCe1 mutations, explore the topology of NBCe1, and-in collaboration with another laboratory-study the biochemistry of the isolated cytoplasmic N termini of NBCe1-A and -B. The proposed work should elucidate the role that the electrogenic Na/HCO3 cotransporter plays in renal function, both in health and disease. The results could have important implications for understanding the normal control of acid-base balance and renal-tubule acidosis.
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