Calcium-mediated agonists are important modulators of intestinal secretory diarrhea acting alone (e.g., rotavirus, Vibrio parahaemolyticus) or in synergism with cAMP-mediated agonists. Indeed, medical costs 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 Ca2+-activated K+ channel, hlK1. As transepithelial Cl- secretion is vectorial in nature it is critical that the ion transporters and channels be correctly targeted to either the apical or basolateral membrane. To date, there is no information regarding the mechanisms by which hlK1 is assembled and trafficked to the plasma membrane in general and to the basolateral membrane in particular. Our long-term goals are to define the molecular motifs responsible for the correct assembly, trafficking, basolateral localization and regulation of hIK1.
In Specific Aim A, we will define the mechanism by which the C-terminal leucine zipper regulates folding and trafficking and regulation of hlK1. Also, we demonstrate that following mutation of the N-terminal leucine zipper, hIK1 fails to traffic to the plasma membrane.
In Specific Aim B we will determine whether the N-terminus acts as a tetramerization domain, whether the N-terminus interacts with the C-terminus and whether mutations affect channel regulation. Also, we will determine whether hlK1 assembles into heterotetramers with other members of the KCNN gene family as well as determine the physiological and pharmacological consequences of this co-assembly.
In Specific Aim C we will define the molecular motifs required for basolateral membrane localization of hIK1 in differentiated epithelia. For these studies we will utilize the MDCK cell line. We will determine the role of adaptors in the localization of hlK1 in general and the epithelial specific mu1B in particular. Finally, we will define the rate of hIK1 endocytosis and determine whether carbachol, insulin and EGF regulate this process. All of our studies will involve a combination of patch-clamp, protein biochemical and immunofluorescence techniques on HEK293, LLC-PK1 and MDCK cells heterologously expressing hIK1. In total, our studies will define, for the first time, the molecular motifs required for the correct assembly, trafficking and localization of a member of the KCNN gene family. As hIK1 plays a critical role in Ca2+-mediated secretory diarrhea and may be a modifier gene of the CF phenotype an understanding of the trafficking and regulation of hIK1 would be expected to be clinically useful
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