Functional Testing of Conserved Regions in Eukaryotic Divalent Metal Transporters

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DESCRIPTION (provided by applicant): Iron is an essential trace element: too little contributes to iron deficiency anemia, while too much iron can lead to organ damage and infection. One way cells control iron levels is by regulating the movement of iron through transmembrane transporters (Nramp2, ferroportin). My preliminary work has identified specific sites in Nramp2 and ferroportin that could be essential to their functions. This project will give me the opportunity to acquire new skills, enabling me to directly test the hypotheses generated by my computational studies. The results may provide key pieces of information (i.e., hepcidin interactions and small molecule inhibition of Nramp2) toward a better understanding of iron transport and regulation. The specific goals are: 1. Test the functional role of conserved amino acid residues in loop E3 of Nramp 1 and Nramp2 (DMT1) through exogenous expression studies and comparative kinetic analysis. 2. Determine metal specificity determinants imparted by loop E3 characteristics. 3. Examine the potential role of loop E3 interactions with iron transport regulators and small molecule transport inhibitors. 4. To continue work on the roles of NRAMP1, NRAMP2, and ferroportin in evolution through bioinformatics methods. PUBLIC HEALTH RELEVANCE: Iron deficiency is a public health problem in the United States and around the world. NRAMP2 is the primary importer of non-heme iron in intestinal epithelium, so a better understanding is needed of its function and regulation.