The long-term objective is the elucidation of the structure and function of Na+-driven amino acid cotransporters. The key to these studies is my recent success in cloning the rabbit and human brush border Na+/glucose cotransporter with functional expression in Xenopus oocytes.
The specific aims are as follows: 1) To clone and sequence intestinal Na+/amino acid transporters such as the intestinal Na+/proline cotransportel,using the expression cloning technique which evolved during the cloning of the Na+/glucose cotransporter. Clones will be sequenced and the deduced primary structures will be used to predict the secondary structures and membrane-spanning regions. 2) To challenge the predictions using biochemical and immunochemical techniques and to analyze the function of the transporters by site-directed mutagenesis and expression of mutants in Xenopus oocytes. Antibodies will be prepared against hydrophilic regions of the transporters. Western analysis, proteolytic experiments, antibody binding studies to brush border vesicles, and glycosylation experiments will be used to probe the secondary structures. Tyrosine and lysine residues of the Na+/proline cotransporter will be mutagenized to assess their possible effects on Na+ and proline binding. 3) To study inherited defects associated with the cloned amino acid cotransporters using Western, Northern and Southern blot analysis.
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