Secretory diarrhea is a prominent feature in intestinal infectious diseases, which are estimated to cost approximately $20 billion in lost productivity in the United States each year. Secretory diarrheas are produced by derangements in the salt and water transport functions of the intestinal mucosa, which are regulated by various hormones, neurotransmitters and immune cell products. Their effects on intestinal cell transport function are mediated by signal transduction pathways involving increases in cyclic nucleotides and Ca. A consensus model of the cellular secretory mechanism in the intestine and other epithelial tissues features regulated Cl channels at the apical (lumen-facing) membranes, and regulated K channels at the basolateral (plasma-facing) membranes of secretory cells. Since the cloning of the cystic fibrosis transmembrane conductance regulator (CFTR), the mechanistic basis of the regulated apical Cl conductance has been clarified, but the K channels responsible for the regulated conductance properties of the basolateral membrane are still poorly understood. Nevertheless, their activity is critical for secretion, and this is a promising site for therapeutic intervention in hyper-secretory states. The central goal of this project is to identify the regulated K channels that participate in the secretory response of intestinal cells. We will define their properties and the regulatory pathways that control their activities. We will attempt to provide structural information that would allow their expression in different tissues and in different pathological states, as well as the regulatory domains that control their activities, to be identified. We will focus our attention on the model intestinal secretory cell lines, T84 and HT29, and use measurements of transepithelial, whole-cell and single-channel currents in epithelial monolayers and cultured cells to identify these channels and study their regulation. In particular, we will focus on K channels activated by the second messengers, Ca and cAMP, K(Ca) and K(cAMP). We will characterize their activation and inhibition by protein kinases and arachidonic acid and their sensitivity to blockers, in order to define the current components activated by signal transduction mechanisms. Finally, we will identify cDNAs encoding intestinal K channels through homology-based amplification of DNA using the polymerase chain reaction with primers corresponding to the conduction pathway of known K channels. This project continues our progress toward defining the mechanisms responsible for intestinal secretion in secretory diarrhea. Based on recent progress in our understanding of the K channels of excitable membranes and renal epithelial cells, we feel that the time is appropriate to reach a similar level of understanding of the K channels of intestinal secretory cells. Pharmacologic agents that affect K channel activity are under intense development. Their application to the intestine could permit therapeutic interventions in life-threatening secretory states.
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