The central goal of this project is to characterize the mechanisms that regulate the Cl and K conductances of salt-secreting epithelial cells. Apical Cl conductance is regulated by agonists (e.g., hormones and neurotransmitters) that use cAMP and Ca as second messengers, and Cl conductance is also increased during cell swelling. These investigators have identified three Cl conductances that can be distinguished from one another based on their biophysical properties and on the effects of blockers. Each of these conductances is activated by a different regulatory pathway (e.g., cAMP, Ca and swelling). The investigators will use whole-cell and single-channel current measurements and flux studies to resolve the specific regulators of each pathway, focusing their attention, in particular, on arachidonic acid metabolites, G-proteins, protein kinase C, calmodulin-dependent kinase and phosphoprotein phosphatases. Using whole-cell current measurements, the investigators will simultaneously collect information on the regulation of basolateral K conductances. The investigators will seek structural information on Cl conductances by expressing specific conductance pathways in Xenopus oocytes from secretory cell RNA. They will use flux studies and double electrode voltage clamp to characterize the expressed conductances and compare them will whole-cell currents recorded from the parent cells. The Cl-secreting human colonic tumor cell line (T84), which exhibits both cAMP- and Ca-dependent Cl secretion, will be used as a model secretory epithelium for the studies. This project will provide a molecular understanding of the conductance pathways that are unique to salt-secreting epithelial cells as well as the cell-specific mechanisms that control their activities.
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