STRUCTURE AND FUNCTION OF ROMK CHANNEL IN KIDNEY

描述：（逐字从应用程序）这5年的赠款申请。IRPG#1， 是三个组成部分赠款的第一个，包括整个IRPG（互动 研究项目补助金）申请。此应用程序还包含单个 核心资源将由各组成部分赠款共享。主要 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， 核苷酸结合结构域和孔形成结构域），和2）使用 ROMK基因敲除小鼠模型研究ROMK通道在肾脏K+调节中的作用 装卸.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提供了 通过磷酸化和核苷酸结合调节通道活性 交互.我们建议检查核苷酸结合和磷酸化， 蛋白质水平。根据我们最近建立的 生产和纯化至均一毫克量的ROMK蛋白的能力。 第一个目标将为我们的合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。的 驱动第二个目标的主要假设是ROMK通道是必不可少的 K+分泌过程在粗升支（TAL）和主要 皮质集合管（CCD）细胞。这一假设将由以下人员进行检验： 使用ROMK基因缺陷的小鼠，即，ROMK“敲除”小鼠模型。 驱动我们选择ROMK“淘汰赛”的关键观察是 证明人ROMK的功能丧失突变产生新生儿 Bartter综合征，一种由于NaCl减少而引起的肾性NaCl消耗性疾病 运输通过TAL。虽然人类ROMK“敲除”证明了 虽然K+通道对TAL功能的重要性，但仍有许多未解之谜 关于这种疾病的病理生理学（例如，持续机制 肾K+损失和前列腺素E2在肾NaC 1和K+消耗中的作用）， 可能最好使用动物模型来解决。

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.