| The epithelial N&barbelow;a+/H&barbelow;+ e&barbelow;xchanger (NHE) isoform, NHE3, is expressed on the apical membrane of the polarized epithelial cells of the kidney and intestine among other tissues, where it functions to allow the absorption of NaCl, NaHCO3, and water. Because of the ease of transfection as well as the ability of directly monitor the activity of the transfected NHE, much of the work characterizing NHE3 has occurred with the protein stably expressed in the exchanger-deficient fibroblast cell lines, PS120 and AP-1. In PS120 cells, it was shown that transfected NHE3 responds to different signaling pathways through discrete domains in its C-terminal tail. In Chapter 1, we examined the regulation of NHE3 by the cationic steroid, squalamine. Squalamine was isolated from dogfish sharks based on its antibacterial properties. Squalamine was also shown to have anti-angiogenic properties and to alter cell volume and shape. Accordingly, we examined whether squalamine regulated the activity of the NHEs: NHE1, NHE2, and NHE3, and determined that squalamine specifically inhibited only NHE3 in a time and concentration-dependent manner, working through the C-terminal 76 amino acids of the protein. In Chapter 2, we examined the role of the C-terminus in the membrane trafficking of the protein. We found that the last 142 amino acids of NHE3 contain at least two independent internalization motifs that ensure a large intracellular pool of the protein, and that the serum-mediated exocytosis of NHE3 occurs independently of the internalization motifs. In Chapter 3, we characterized NHE3 in the more physiological context of a proximal tubule-derived cell line, OK cells. We found that in OK cells, NHE3 is largely intracellular in a recycling endosome compartment. The small fraction of NHE3 that is expressed on the cell surface is detergent-insoluble and in a large molecular weight complex in the center of the cell. |
