Na+ reabsorbing renal epithelia contain Na+ specific channels situated within their apical membranes. These epithelial Na+ channels mediate entry of Na+ from the luminal fluid into the cell during the first stage of electrogenic transepithelial Na+ transport. Restriction of the Na+ channels to the apical membrane domain is essential for the vectorial transport of Na+ across these epithelia. However, the mechanisms responsible for determining and maintaining the distribution of the Na+ channels to the apical membrane domains of renal Na+ reabsorbing epithelia are presently not understood. Preliminary experiments suggest that epithelial Na+ channels are linked to the membrane cytoskeletal proteins ankyrin and spectrin in renal epithelia. It is hypothesized that this interaction between Na+ channels and the membrane cytoskeleton is involved in the maintenance of the polarized distribution of Na+ channels to the apical membrane. Thus, the primary goal of this proposed study is to investigate the role of the membrane cytoskeletal proteins ankyrin and spectrin in determining and maintaining the apical cell surface distribution of epithelial Na+ channels in Na+ reabsorbing renal epithelia. A6 renal epithelial cells, a cell line derived from the distal tubule of Xenopus laevis will be used as the model system. There are three specific aims:(1) To test the hypothesis that there is a direct interaction between epithelial Na+ channels and the membrane cytoskeletal proteins ankyrin and spectrin, and that this interaction restrict the Na+ channels to the microvillar domain of the apical membrane in Na+reabsorbing renal epithelia using biochemical, immunocytochemical and fluorescence photobleach recovery (FPR) techniques (2) To test the hypothesis that Na+ channel-membrane cytoskeleton interactions are involved in determining the distribution of epithelial Na+ channels to the apical membrane of Na+ reabsorbing renal epithelia by examining differentiation from unpolarized precursors to polarized epithelia cells in A6 cells. (3) To further test the hypotheses that the Na+ channel- membrane cytoskeleton complex identified in Specific Aim 1 is involved in determining and maintaining the distribution of Na+ channels within specific membrane domains, the Xenopus oocyte expression system will be used as an in vitro model. This will be achieved by injecting oocytes with Poly (A)+RNA coding for the epithelial Na+ channel or coinfection of Na+ channel Poly (A)+RNA with either sense or antisense oligonucleotides encoding ankyrin followed by immunochemical and FPR analyses of expressed channels. In addition to elucidating how the apical distribution of this essential channel protein is determined and maintained in renal epithelia, the studies proposed in this application will increase our understanding of how other epithelial ion channels are established and maintained within their respective membrane domains. Furthermore, in light of the recent evidence suggesting that ischemic injury disrupts the membrane cytoskeleton of the renal tubules and their ability to maintain distinct apical and basolateral membrane domains, our studies will increase our understanding of ischemia induced renal failure.
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