Calcium-mediated agonists are important modulators of intestinal secretory diarrhea acting alone (e.g., Vibrio parahaemolyticus toxin) or in synergism with cAMP-mediated agonists. Medical costs associated with infectious diarrhea are estimated at $23 billion annually in the United States. A critical step in the Cl-secretory process is the activation of a basolateral membrane Ca/2-activated K+ channel (K/Ca). Thus, our long term goals are to understand the physiological regulation of this K/Ca as well as to clarify the role of pharmacological modulators of K/Ca in disease. There is a clear dissociation between the Cl-secretory response and intracellular Ca/2+ during Ca/2+-mediated Cl- secretion suggesting that second messengers other than Ca/2 are important modulators of this K/Ca. We demonstrate that the recently cloned intermediate conductance K/Ca (hIKl) os expressed in both human colonic and airway epithelial and this channel is activated by PKA-dependent phosphorylation. The first major aim of this proposal is to define the mechanism whereby phosphorylation of hIKl modulates its Ca/2+- dependent regulation. We will study hiKl heterologously expressed in Xenopus oocytes using both two-electrode voltage-clamp (TEVC) and excised patch-clamp techniques. We will mutate the single PKA phosphorylation consensus site to demonstrate that phosphorylation of this serine is critical in modulating the Ca/2+-dependent activation of hIKl. We will also study endogenous hIKl in colonic and airway epithelia, using excised patch- clamp techniques, to determine which phospatases are important in dephosphorylating hIKl. These studies will allow for a complete understanding of how increasing cellular cAMP modulates K/Ca and hence the Cl-secretory response associated with diarrhea involving cAMP- and Ca/2+-dependent agonists. Clotrimazone is a potent blocker of hIKl in colonic epithelia, suggesting that clotrimazole may be useful as an antidiarrheal. Also, clotrimazone is known to block the Gardos channel in red blood cells. As inhibition of the Gardos channel prevents RBC sickling, clotrimazone is being evaluated for clinical efficacy in the treatment of sickle cell anemia. Indeed, it appears as the Gardos channel of RBCs is hIKl. Thus, the second major aim of this proposal is to define, at a molecular level, the mechanism by which clotrimazone inhibits hIKl. We have narrowed the binding site down using chimeric constructs between hIKl and the related SK channels. We will construct additional chimeras before using point mutations to define the amino acids which determine the clotrimazone binding site. These studies will be carried out using TEVC and excised patch-clamp techniques. A complete understanding of the mechanism of clotrimazone block will be important for the treatment of sickle cell anemia and, potentially, diarrhea.
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