MOLECULAR BASIS OF RENAL EPITHELIAL CYSTINE TRANSPORT

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Cystinuria, one of the most common genetic disorders, is characterized by excessive excretion of cysteine and basic amino acids in urine. The low solubility of cysteine results in formation of kidney stones which can eventually lead to renal failure. The disease is due to defects in a high- affinity transport system for cysteine in the brush border membranes (BBMs) of kidney and intestinal epithelial cells. The molecular properties of proteins involved in epithelial cystine transport are incompletely understood. A protein (NBAT), cloned in this laboratory from rat kidney and shown to be localized in the renal and intestinal BBMs, has been implicated in this transport. Although mutations in human NBAT gene have been found in one form of cystinuria, there are reasons to believe that defects in other, as yet uncharacterized, genes may also be involved. Recently, this laboratory showed that kidney and intestinal NBAT is associated with another protein (denoted P50) and proposed that the heterodimer represents the functional unit of the high-affinity cystine transporter in these membranes. The Specific Aims of this proposal are to clone and characterize P50 and elucidate its role in epithelial cystine transport. The methods proposed to achieve these objectives include purification and eventual cloning of P50 from rat kidney. Site-specific antibodies will be used for the molecular characterization of the protein and for its tissue and cellular localization. The Xenopus oocyte expression system will be used to investigate the relative role of P50 and NBAT in amino acid transport. If necessary, other expression systems (eg. COS7 and CHO cells) will also be employed. The proposed research is expected to enhance knowledge of the molecular mechanisms involved in epithelial cystine transport and, eventually, lead to a more complete understanding of the molecular and genetic factors responsible for producing the cystinuric syndrome.