A major portion of the studies proposed will be carried out using two recently cloned human peptide transporters, hPEPT1 and 2. They will involve functional expression in two different heterologous systems (vaccinia virus for expression in mammalian cells and Xenopus oocyte expression system) and detailed characterization of their functional and operational mechanisms. Function will be monitored by measuring the transport of radiolabeled substrates as well as by quantifying peptide-induced membrane currents using the two microelectrode voltage-clamp technique. Several individual deletion mutants and PEPT1-PEPT2 chimeras will be generated and used in the functional studies to identify the substrate binding domains in the transport proteins. The potential roles of the PEPT1 and 2 transporters in intestinal and renal absorption of zinc, copper and other micro nutrients as peptide-metal chelates will be explored using the cloned transporters, cultured intestinal and renal cell lines that express the two transporters differentially and also isolated intestinal cells and renal brush border vesicles. Functional expression and localization (cell types and membrane domains) of the transporters in the liver and the pancreas, where there is compelling evidence for their expression, will be studied. Regulation of transporter activity and expression will be examined in cultured cells. These studies are well justified by the clinical, nutritional and pharmacological importance of peptide transporters in humans and will provide a better understanding of the physiological, biochemical and molecular aspects of these transporters.
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