NBCe1 and n1: Crystal Structure Determination

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DESCRIPTION (provided by applicant): Bicarbonate (HCO3-) transport across epithelial cells of the kidney is of great importance to maintain systemic acid-base balance. The HCO3- transporters NBCe1 & NBCn1 are integral, membrane-proteins that, respectively, play important roles in HCO3- reabsorption in the proximal tubule and NH4+ reabsorption in the thick ascending limb. The specific aims of this project are: (1) To use X-ray crystallography to illuminate the structures of the N-terminal domains of NBCe1 and n1; (2) To explore the physiological roles in NBCn1 of the unique 124-residue exon 7, which may be spliced in or out of the protein; (3) To develop a large-scale, eukaryotic expression system for the membrane domain of NBCe1 or NBCn1, to be used later for crystallization trials. My preliminary work on the purification and crystallization of the N-terminus of NBCe1 is paving the way for vital structural information needed to study the regulation of HCO3- transport, and my new splice variants of NBCn1 broadens our current understanding of the role of NBCn1 in various cell types. The proposed study will provide key insights on the molecular mechanisms of renal HCO3- transport and will enhance our understanding of processes fundamental to renal disease.