In the past, studies of epithelial transport have focused largely on the active transport pathway through epithelial cells. The transport pathway between cells, the paracellular pathway, is now receiving increased attention. But no epithelium displays the dynamic regulation of paracellular transport as well as Malpighian (renal) tubules of the blood-feeding mosquito Aedes aegypti. Here, the diuretic hormone leucokinin triggers a 10-fold increase in paracellular Cl conductance that helps rid the mosquito of the unwanted plasma fraction of the blood meal. The effects of leucokinin are reversible, and switch like on/off changes suggest the post- translational modification of paracellular proteins. At the level of Malpighian tubules, leucokinin binds to a G protein-coupled receptor that leads to Ca entry into the cell via Ca channels activated by Ca store depletion. How Ca goes on to increase the Cl conductance of the septate junction (functional equivalent of the vertebrate tight junction) is unknown. Furthermore, the proteins that define the conductance and the permselectivity of septate junctions are largely unknown. Accordingly, one goal is to identify the septate junctional proteins in Malpighian tubules of A. aegypti by the methods of proteomics. The proteomic analysis will focus on plasma membrane proteins that may extend into the paracellular space to define paracellular conductance and permselectivity. Physiological/pharmacological studies in intact Malpighian tubules seek to uncover the Ca-activated kinases, phosphatases, and/or G-proteins that mediate the rapid paracellular conductance changes, and proteomic analyses will attempt to identify those septate junctional proteins undergoing posttranslational modifications with the rapid on/off effects of leucokinin. Time permitting, the goal of molecular cloning is to confirm the proteomic identification of septate junctional proteins, and newly produced antibodies seek to examine whether identified septate junctional proteins are indeed located at the septate junction of Malpighian tubules. While identifying new proteins of the invertebrate septate junction, the results may reveal how kinases and other enzymes regulate paracellular transport, thereby integrating transcellular and paracellular transport pathways with additive if not synergistic effect. A similar integration may well take place in vertebrate epithelia where hormones and neurotransmitters are known to trigger quick and potent responses in secretory epithelia such as salivary glands, stomach, pancreas and intestine. ? ? ?
