STUDIES ON BASOLATERAL MEMBRANE POTASSIUM CHANNELS

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The long-term goals of the proposed research project are logical extensions of our efforts during the past few years and are designed to further our understanding of the regulation and structure-function relations of two K+ channels that are present, in basolateral membranes of intestinal cells and that have been successfully reconstituted into planar phospholipid bilayers. The first is an "inwardly rectifying K+ channel that is present in basolateral membrane vesicles isolated from Necturus small intestinal cells. The second is a Ca-activated, high ("maxi") conductance channel that is present in basolateral membrane vesicles of rabbit colonic epithelial cells. The five specific aims of the proposed research program are: I. To examine the effects of possible intracellular mediators and/or second messengers on the activities of these channels reconstituted into planar phospholipid bilayers. II. To purify these channel proteins in functional forms as determined by the ability to reconstitute their single channel activities in planar bilayers. Preliminary studies to be described below have disclosed feasible approaches towards that goal. III. To clone the cDNAs encoding these proteins and determine their primary structures (i.e. amino acid sequence) and putative "membrane topologies" using Kyte-Doolittle hydropathy analyses. IV. To express these channels in Xenopus oocytes using the poly(A+)mRNAs derived from the cloned cDNAs to confirm that we have in fact cloned the correct cDNAs and open the avenue for the study of structure-function relations using site-directed mutagenesis. V. To confirm the putative membranes of origin and explore the tissue distribution of these channel proteins using immunohistochemical techniques.