The long term goal of this program is to examine the control and regulation of ion transport in epithelial tissue. In particular this project will use single channel and biochemical methods to examine the regulation of amiloride-blockade sodium channels in renal and lung epithelial cells. These channels are interesting because of their relative uniqueness among channels in transporting tissue and because of the interesting hormonal regulation of these channels. However, the mechanisms for regulation of these channels have not been completely described. Therefore, this project will further investigate the signaling cascades which regulate sodium channels in three sodium-transporting epithelial cell lines using patch clamp techniques supplemented by direct biochemical measurements.
The specific aims for the proposed grant period will investigate four signaling cascades that regulate sodium transport.
The aims are (1) further examine the regulation of sodium channels by heterotrimeric G protein signaling cascades; specifically, what is the nature of the interaction between Galpha-3 and ENAC; do the G protein alpha subunits activate Na channels directly or do they activate some other effector molecule closely associated with the inner surface of the apical membrane; and do G protein betagamma subunits alter ENaC activity? (2) Examine the regulation of sodium channels by small G protein signaling cascades. The activation of one small G protein, K-Ras2A, is required to sustain normal ENaC activity. Elements of the K-Ras signaling cascade appear to be closely associated with the cytosolic surface of the apical membrane since the cascade can be activated in excised, inside-out patches. Therefore, the mechanism of activation of K-Ras and the signaling molecules activated by K-Ras will be examined. 3) Examine the regulation of sodium channels by inositol lipids and inositol lipid kinases. Sodium channels in excised, inside-out patches require the presence of phosphatidylinositol-4,5-bis-phosphate (4,5-PIP/2) and A6 cells have the necessary enzymes to produce 4,5- pip/2. (4) Investigate the mechanisms by which aldosterone increases sodium channel activity. Demonstrate that the signaling cascade that begins with aldosterone activation of K-Ras and leads to the PI-3K- mediated production of 3,4,5-PIP3 involves activation of phosphatidylinositol-dependent kinase (PDK1/2), serum glucocorticoid- dependent kinase (SGK), and the ubiquitin ligase, Nedd4. Determine that these signaling molecules are activated by activation of PI-3-kinase and that 4-PIP-5-kinase is activated to produce 4,5-PIOP2 and subsequently 3,4,5-PIP/3. Finally, we will use commercially available gene chips to identify new aldosterone-induced genes.
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