MEMBRANE PROCESSES MEDIATING NACL TRANSPORT

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The overall goal of this project is to understand the mechanisms by which the reabsorption of salt from the urine by the kidney is controlled, particularly in the distal nephron where hormonal signals regulate Na reabsorption according to the needs of the body. The specific goals of this application can be put into two general categories. The first is a detailed description of the regulation by the adrenal mineralocorticoids of ion transporters in the cortical collecting tubule of the rat kidney. The second is an exploration of the mechanisms by which aldosterone controls the activity of epithelial Na channels. To achieve the first goal, we will use the patch clamp technique to monitor channel and pump-mediated currents across the entire cell membrane (whole-cell recordings) as well as through individual channels (single-channel recordings). The time course and the extent of activation of apical Na channels, apical K channels, and basolateral Na/K pumps will be evaluated in vitro after manipulations designed to effect the mineralocorticocoid status of the animals in vivo. These manipulations include changes in diet (low Na, high K), Na depletion using diuretics such as furosemide, and direct infusion of steroids. Specific issues to be addressed include (1) Are changes in channel activity accounted for by changes in levels of plasma aldosterone? (2) changes in overall transport activity accounted for by changes in the number of conducting channels? (3) Are channels expressed evenly over the membrane or in clusters? (4) Are individual cells stimulated in a graded or and all-or-none fashion? and (5) Are changes in channel activity accompanied by parallel changes in activity of the NA/K pumps? The second set of aims will test several specific hypotheses concerning the mechanisms through which Na channels are regulated. These are (1) aldosterone activates pre-existing channels without stimulating channel MRNA synthesis, (2) aldosterone promotes lipid or protein methylation leading to activation of channels, (3) aldosterone activates Na/H exchange leading to alkalinization of the cytoplasm, and (4) ADH, acting through CAMP, promotes the fusion of vesicles containing aldosterone- activatable channels with the apical membrane. These hypotheses will be tested using patch-clamp electrophysiology, fluorescence measurements of cell Ph, and expression of channels in oocytes by injection of MRNA. The proposed work should enhance our understanding of processes that regulate body NaCl content in health and which may be defective in disease states such as cystic fibrosis and hypertension.