The research is aimed at understanding small intestinal chloride transport at the brush border and molecular level. We have recently identified as the major chloride transport pathways across the brush border membrane: 1) chloride/hydroxide exchange and 2) chloride conductance. Both are sodium independent. Taking advantage of a new conceptual framework for quantitating transport in membrane vesicles, we now propose to separately determine the chloride/hydroxide exchange activity and the chloride conductance in jejunum, ileum, villus tip and base cells, as well as after induction of a secretory state with cholera toxin. The studies should provide information on the contribution of the membrane to the electrolyte transport properties of the intact tissue in different regions and different absorptive or secretory states. To better understand the molecular properties of the transporters, chloride transport activity will be measured in vitro in isolated membranes and its dependence determined on pH, temperature, and calcium concentration. In addition, specificity for other anions besides chloride will be evaluated, bicarbonate is of particular interest. Attempts will be made to isolate the proteins responsible for chloride/hydroxide exchange and to reconstitute functionally intact chloride transport in liposomes. To facilitate biochemical as well as cell physiological studies, antisera against SITS binding proteins, which include the exchange transporter, will be produced from the brush border membrane.
