The main goal of the proposed research is to understand at the molecular level the mechanisms and regulation of ion flow through conductive Na+ entry channels that exist in the outer (or apical) plasma membrane of most Na+ reabsorbing epithelia. This Na+ entry channel is rate-limiting for overall transepithelial Na+ reabsorption, and is regulated hormonally, specifically by the peptide hormone vasopressin and the steroid hormone aldosterone. This entry process is passive, and is sensitive to inhibition by the diuretic drug amiloride. Experiments are designed to test the hypothesis that the protein isolated from A6 cells and bovine kidney functions as an amiloride-sensitive ion channel. There are five specific aims: 1) To analyze the kinetic characteristics of this purified putative Na+ channel protein, utilizing reconstitution procedures to measure single channel characteristics in planar lipid bilayers. These experiments will elucidate the cation selectivity of the channel, the nature, the sidedness, and voltage dependence of the amiloride block, and whether phosphorylation or methylation of the protein per se has any functional consequence. 2) To determine biochemically whether vasopressin and aldosterone regulate Na+ channels by examining whether the increased density of functional Na+ channels results from a recruitment of channels from a cytoplasmic pool or by activation of quiescent channels already resident in the membrane. 3) To examine the contribution of the cortical actin filament network and its associated proteins to the regulation of epithelial Na+ channel activity. 4)To clone and sequence full-length cDNA's of each subunit. Once accomplished, polyclonal antibodies against each subunit will be prepared in order to investigate the topological arrangement of the subunits within the apical membrane. Using cDNA probes and polyclonal antibodies, the regulation of messenger RNA and protein expression of the different subunits by vasopressin and aldosterone in renal A6 cells using Northern blot analysis, ribonuclease protection assays, and Western blot analysis will be studied. 5) To determine the functional role of each subunit in amiloride-sensitive Na+ transport in sense and anti-sense oligonucleotide experiments. These studies will further our knowledge of the physiological, biochemical, and molecular biological properties of this ubiquitous transport system, and increase our understanding of the mode of action of amiloride and other diuretic compounds.
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