STRUCTURE AND FUNCTION OF ROMK CHANNEL IN KIDNEY

描述：(逐字摘自申请表)这份为期5年的拨款申请。IRPG#1， 是构成整体IRPG(互动)的三个组成部分中的第一个 研究计划资助)申请。此应用程序还包含单个 核心资源.HAT将在组件赠款之间共享。少校 IRPG#1的目标是1)定义ROMK上的功能结构域 参与通道调节的K+通道(磷酸化位点， 核苷酸结合域和孔形成域)，以及2)使用 ROMK基因敲除小鼠模型中ROMK通道在肾脏K+中的作用 正在处理。驱动第一个目标的假设是氨基和 ROMK通道蛋白的羧基末端区域为 磷酸化和核苷酸结合对通道活性的调节 互动。我们建议研究核苷酸结合和磷酸化在 蛋白质水平。这现在是可能的，基于我们最近建立的 能够生产和纯化均一毫克量的ROMK蛋白。 第一个目标将为我们的协作提供必要的关键信息 来获得这个K+通道的三维结构信息。这个 推动第二个目标的主要假设是ROMK通道是必不可少的 粗大升肢(TAL)和主干的K+分泌过程 皮质集合管细胞(CCD)。这一假设将通过以下方式检验 使用ROMK基因缺陷的小鼠，即ROMK“敲除”小鼠模型。 促使我们选择ROMK“淘汰赛”的关键观察是 人类ROMK功能缺失突变导致新生儿的证据 巴特综合征，一种由于NAC1减少而导致的肾脏NACI耗竭情况 由TAL运送。而人类ROMK的“击倒”则展示了 K+通道对TAL功能的重要性，仍有许多悬而未决的问题 关于这种疾病的病理生理学(例如，持续的 肾脏K+丢失和前列腺素E_2在肾脏NaC_1和K~+消耗中的作用 最好是用动物模型来解决。

DESCRIPTION: (verbatim from application) This 5-year grant application. IRPG#1, is the first of three Component Grants comprising the overall IRPG (Interactive Research Project Grant) application. This application also contains the single CORE of resources .hat will be shared among the Component Grants. The major goals of IRPG#1 are 1) to define the functional-structural domains on the ROMK K+ channel that are involved in channel regulation (phosphorylation sites, nucleotide-binding domain and pore-forming domain), and 2) to determine using a ROMK knockout mouse model the functional roles of the ROMK channel in renal K+ handling. The hypothesis driving the first goal is that amino- and carboxy-terminal regions of the ROMK channel protein provide domains for regulation of channel activitv by both phosphorylation and nucleotide-binding interactions. We propose to examine nucleotide binding and phosphorylation at the protein level. This is now possible based on our recently established ability to produce and purify to homogeneity milligram amounts of ROMK protein. The first goal will provide crucial information necessary for our collaboration to obtain three-dimensional structural information on this K+ channel. The major hypothesis driving the second goal is that the ROMK channel is essential to K+ secretory processes in the thick ascending limb (TAL) and in principal cells of the cortical collecting duct (CCD). This hypothesis will be tested by using mice deficient for the ROMK gene, i.e., a ROMK "knockout" mouse model. The key observation driving our choice of the ROMK "knockout" is the demonstration that loss-of-function mutations of human ROMK produce neonatal Bartter's Syndrome, a renal NaCI wasting condition due to reduced NaC1 transport by the TAL. While the human ROMK "knockout" demonstrates the importance of this K+ channel to TAL function, many unanswered questions remain concerning the pathophysiology of this disorder (e.g., mechanism for continued renal K+ loss and role of prostaglandin E2 in renal NaC1 and K+ wasting) and may best be addressed using an animal model.