REGUALTION OF A NOVEL CGMP-GATED K+ CHANNEL IN KIDNEY

环核苷酸门控钾通道的分子结构 （Knuc）仍然没有定义。 Knuc是K通道的重要类别， 它可能参与动脉张力的调节， 在高血压的发病机制中起重要作用。 基于 系统发育数据，我们假设Knuc应该包含基本的 两种Shaker（编码电压门控K通道的基因家族）的特征 K通道和环核苷酸门控非选择性阳离子（Cnuc） 渠道 一种兔探针，其衍生自 分离cGMP门控阳离子通道。 该基因编码一种新的 多肽（Kcn），含有电压的基本特征- 门控K通道 最重要的是，不像其他任何振动台K通道 蛋白质，Kcn含有一个环核苷酸结合结构域。 爪蟾 注射Kcn RNA的卵母细胞表达电压门控性钾电流， 由cGMP激活。 据我们所知，这是第一个 cGMP门控K通道的描述。 KCN可以，部分地， 增加细胞内cGMP的血管舒张物质的作用。 基于上述发现，我们建议进一步表征Kcn 蛋白质的动力学性质（在非洲爪蟾中的表达 卵母细胞）和组织分布（免疫细胞化学）。 的细胞系 将产生稳定转染Kcn基因的细胞， 用于检查Kcn蛋白调节的几个方面：Kcn是 由物质调节的功能（心房利钠肽（ANP）， 血管加压素（AVP）等），增加细胞内cGMP和cAMP， 培养细胞？ 蛋白质在体内和体外是否被磷酸化？ 哪种激酶最能解释激素（AVP，AV）对 渠道功能？基因表达是糖皮质激素反应性的吗？ 使用 定点诱变，我们还将解决有限数量的 关于环的结构-功能关系的问题 核苷酸结合位点和推定的磷酸化位点。

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.