The molecular structure of cyclic nucleotide-gated potassium channels (Knuc) is still undefined. Knuc is an important class of K channel since it may participate in the regulation of arterial tone and therefore play an important role in the pathogenesis of hypertension. Based on phylogenetic data, we hypothesized that Knuc should contain the essential features of both Shaker (gene family encodes voltage-gated K channels) K channels and of cyclic nucleotide-gated non-selective cation (Cnuc) channels. A rabbit probe derived from the nucleotide binding domain of cGMP-gated cation channels was isolated. The gene encodes a novel polypeptide (Kcn) that contains the essential characteristics of voltage- gated K channels. Most importantly, unlike any other Shaker K channel protein, Kcn contains a cyclic nucleotide-binding domain. Xenopus oocytes injected with Kcn RNA express a voltage-gated K current that is activated by cGMP. This is to our knowledge the first molecular description of a cGMP-gated K channel. Kcn could, in part, mediated the effects of vasodilatory substances that increase intracellular cGMP. Based on the above findings, we propose to further characterize the Kcn protein in terms of its kinetic properties (Expression in Xenopus oocytes) and tissue distribution (Immunocytochemistry). A cell line that is stably transfected with the Kcn gene will be generated and will be used to examine several aspects of Kcn protein regulation: Is Kcn function regulated by substances (Atrial natriuretic peptide (ANP), Vasopressin (AVP) etc) that increase intracellular cGMP and cAMP in cultured cells? Is the protein phosphorylated in vivo and in vitro? Which kinases best explain the observed effect of hormone (AVP, AV) on channel function? Is gene expression glucocorticoid-responsive? Using site directed mutagenesis, we will also address a limited number of questions regarding structure-function relationships of the cyclic nucleotide binding site and the putative phosphorylation sites.
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