Na/H exchangers (NHE) control the movement of salt, water and acid-base equivalents, and play a critical role in pH regulation, cell proliferation, volume control and ion homeostasis. A wide range of clinical conditions are associated with derangement's in Na/H exchange, including hypertension, cardiac ischemia and various acid-base disorders. Although a number of plasma membrane NHE isoforms have been described in higher vertebrates, endosomal Na/H exchangers represent a physiological important sub-type that have yet to be identified at the molecular level. The-yeast NHE homologue, Nhx1, has been cloned and localized to a unique, late endosomal compartment, thus providing a starting point to explore the cellular and physiological role of intracellular Na/H exchange. Using a fully functional, epitope-tagged transporter and an array of secretory and trafficking mutants the cellular routing of Nhx1 from synthesis to degradation will be characterized. Further clues on function will derive from localization studies following exposure of cells to salt, osmotic stress and pH perturbations. Ion transport activity will be characterized in purified vesicles with respect to the reversibility of Na/H exchange, selectivity and affinity for ions, and sensitivity to a range of inhibitors, including the diuretic amiloride and its derivatives. These studies will form the foundation of studies designed to map the structure and function of the exchanger. In addition, NHX1 gene expression in response to a range of physiological signals and search for protein factors interacting with putative regulatory domains in promoter regions, and at the C-terminus of the exchanger. These approaches may lead to a better understanding of the signal transduction pathways involved in cell volume regulation, hypertonic response and pH homeostasis. Expression and localization of the human homologue of the yeast Nhx1 will serve as a starting point for understanding the function of a putative endosomal mammalian NHE.
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