The rate of epithelial fluid secretion is controlled by the activity of apical membrane Cl- channels. Defective regulation of these channels results in potentially lethal diseases. This project will investigate a novel mechanism controlling the functional expression of epithelial Cl- channels. Hydrophobic affinity chromatography can be used to isolate, from Cl- secreting epithelia, a unique intracellular membrane fraction, designated Mps, enriched in a single type of Cl- channel. This same channel is found in the apical membrane, as well. Recent studies identify a 200kDa protein that copurifies with the Mps as nonmuscle myosin IIA and the origin of the Mps as the trans-Golgi network (TGN). The long range goal of this project is to test the hypothesis that the Mps represents a vesicular intermediate that shuttles Cl- channels into and out of the apical membrane to regulate the rate of epithelial electrolyte and fluid secretion. The biochemical explanation for Mps adherence to hydrophobic resins is unclear and understanding the mechanisms governing this phenomenon could provide significant insights into the cellular regulation of Mps function. The same chromatographic methods are used to isolate calcium-binding proteins, including the annexins, suggesting that these proteins may be mediating Mps binding to hydrophobic column resins. Therefore, the specific aims of this project are to identify and partially purify the annexins expressed by the tissues and cells from which Mps is isolated. Then, these partially purified components will be used to try and reconstitute Mps binding to hydrophobic resins. An additional aim is to test whether the Mps functions to ferry newly synthesized secretory proteins from the TGN to the apical membrane of cultured epithelia. Pursuit of these specific aims will help define the function of Mps and provide essential preliminary data for the development of a more broad project into the role of Mps in the cellular and molecular control of epithelial fluid and electrolyte secretion and the biogenesis of structural polarity in epithelial cells.
