Malpighian Tubule Transport Physiology: Mechanisms and Regulation

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The overall goal of this project is to identify and characterize current-generating and current-dissipating transport systems in the natural environment of an intact principal cell in the Malpighian tubule of the yellow fever mosquito (Aedes aegypti). In the basolateral cell membrane, the aim is to characterize K channels, and putative Na and Ca channels. In the apical membrane, the focus is on current generation by the V-type H+-ATPase, testing the effects of voltage and the two substrates of the pump, H and ATP. Investigating how current is dissipated across this membrane may lead to the discovery of a new family of antiporters.Outside principal cells, the grant proposes to characterize the nature of the shunt taken by Cl during transepithelial secretion. One hypothesis to be tested is that the stellate cell serves as the shunt pathway. The alternate hypothesis considers insect septate junctions to offer a paracellular pathway for Cl. They hypothetical model proposes that septate junctions house a Cl channel-like structure that is regulated by the diuretic peptide leucokinin.Experiments described in this proposal will demonstrate how proton pump, channels, symporters and antiporters are organized in principal cells in insect Malpighian tubules to secrete Na and K into the tubule lumen via a transcellular path. Studies of the shunt pathway will show how Cl is secreted, presumably via a paracellular path. Together, these studies will reveal the combinatorial arrangement of the molecular building blocks of epithelial transport: pumps, channels, carriers, and junctions and how they interact to secrete NaCl, KCl, and water into the tubule lumen. Results from the proposed studies of an ATP-driven pump in an intact cell will be of wide interest to biologists.