The proposed 5-year extension of the MERIT Award will focus on the completion or evolution of five long- term projects that began during the initial 5-year period. The first three are expansions of the original three Specific Aims. (1) Molecular tools and localization studies: (1 a) Complete characterization of new NBCe2 antibody. (1b) Determine localization of NBCe2. (1c) Characterize new splice variants of NBCe2, which plays a critical role in the choroid plexus (kidney of the brain). (1d) Complete the creation of a conditional NBCe1-null mouse. (2) Properties of wild-type electrogenic NBCs: (2a) Use Biacore to rule out CA II- NBCel interactions with a rapid off rate, and test graded [HCO3]o levels in oocytes expressing NBCel ad CA IV to uncover potential CA IV stimulation at extremely low [HCO3]o levels. (2b) Extend CO3= project by determining effect on inward currents of systematically varying [NaCO3-]o. (2c) Use giant inside-out patch in conjunction with out-of-equilibrium CO2/HCO3 solutions to determine pHi dependence of NBCel. (3) Structure-function analyses: (3a) Continue analysis of natural disease-causing human mutations. (3b) Complete DIDS knock-off project. (3c) Complete the characterization of the DIDS-binding site on outer side of TM12. (3d) Use inside-out giant patches to explore cytoplasmic DIDS-binding motifs. (3e) Complete on- going study to determine the role of the four conserved Cys residues in 3rd extracellular loop of NBCel. The last two projects began during the current initial 5-year period: (4) NBCe2 binding partners., (4a) Systematically characterize interactions between NBCe2 and potential binding partners using cell bio-logical and cell physiological approaches. (4b) Determine whether any of the interactions shifts the Na:HCO3 stoichiometry of NBCe2 from 1:2 to 1:3. (5) Structural biology: (5a) complete the model-building stage of the determination of the X-ray crystal structure of the large cytoplasmic N terminus of the renal splice variant NBCe1-A. (5b) Solve the crystal structure of the larger cytoplasmic N terminus of the ubiquitous splice variant NBCel-B. The proposed work should elucidate the role that the electrogenic Na/HCO3 cotransporters play in renal function and acid-base homeostasis, both in health and disease. The results could have important implications for understanding the normal control of acid-base balance and renal-tubule acidosis.