In mammals, urea is the predominant end-product of nitrogen metabolism and plays a central role in the urinary concentrating mechanism. Regulation of urea excretion and accumulation in the renal medulla depends on the functional state of vasopressin-sensitive, phloretin-inhibitable urea transporters. We have recently isolated cDNAs encoding the urea transporters from rabbit (UT2) and rat kidney medulla (rUT2) as well as from human erythrocytes (HUT3). The objective Of this proposal is to elucidate the structure, function and regulation of expression of urea transporters. To identify regions in their primary sequences responsible for urea translocation, phloretin inhibition and sensitivity to mercurial reagents we propose to modify these proteins by genetic engineering and to study the mutant proteins by expression in Xenopus oocytes. We propose to construct chimeras between UT2 and HUT3 in order to assign functional properties to specific regions of UT2 and HUT3 and to used site-directed mutagenesis to pinpoint individual amino acid residues involved in passive urea transport. To model the urea binding site we propose to employ a novel approach involving capillary electrophoresis which will allow us to determine the effect of specific mutations on substrate-specificity. A scanning mutagenesis program that exploits the reactivity of cysteines placed at strategic locations within the molecule to PCMBS will be used to probe the translocation pathway of these proteins. To study the secondary structure of urea transporters we will attempt to develop an overexpression system in E.coli and use the purified and reconstituted protein for spectroscopy and to perform crystallization trials. To determine whether vasopressin-induced activation of transport in kidney involves recruitment of urea transporters from intracellular vesicle pools of terminal IMCD cells we propose to study the expression of rUT2 in intracellular vesicles by immunogold electronmicroscopy and to determine whether acute treatment with vasopressin triggers insertion of rUT2 molecules in the plasma membrane. Since we demonstrated that rUT2 mRNA levels are highly responsive to changes in the hydration state of the animal or to protein dietary manipulations we propose to determine the mediators which cause these responses and to study the mechanism underlying differential regulation of the 2.9 and 4.0 kb transcripts of rUT2. The results from these studies should advance our understanding of the mechanisms that enable mammals to concentrate urine and should provide information of general importance to our understanding of how facilitated solute transporters function.
