Structure and Function of ROMK Channel in Kidney

描述（由申请人提供）：ROMK （Kir1.1）在远端肾元和收集管中形成顶端K通道，为1)粗升肢吸收NaCI所需的K循环和2)连接管道和主细胞分泌K提供K分泌途径。因此，定义ROMK通道的功能和调控对于理解肾钾处理和体内钾稳态非常重要。ROMK的功能丧失突变导致Bartter综合征，这是一种低血压的肾盐消耗疾病，是由于粗大的升肢丧失了盐的再吸收能力。我们开发出了唯一的ROMK Bartter’s小鼠，具有足够高的存活率（>30%），可以进行生理学研究，也是唯一具有如此高存活率的Bartter’s小鼠模型。我们建议继续研究小鼠ROMK Bartter's的病理生理学，以提高我们对ROMK在肾脏K处理中的作用的理解，并提出治疗人类Bartter综合征的潜在方式。我们还将开发两种新的ROMK转基因小鼠，它们将为评估ROMK异构体的特定作用和ROMK异构体在天然肾细胞中的调节运输提供模型。其中一种异构体ROMK1仅在远端小管晚期和集管中表达，并被认为在膳食K摄入量调节肾K分泌中发挥重要作用。我们建议使用Cre-LoxP策略通过选择性删除ROMK1特异性外显子来产生ROMK1缺陷小鼠。我们假设这只小鼠不会有Bartter的表型，相反，随着饮食中K摄入量的增加或减少，K处理失调。我们还将生成带有FLAG和EGFP标记的ROMK1 bac转基因小鼠，以确定ROMK在肾上皮细胞中的运输，并确定ROMK调节复合体中的相关蛋白。这些小鼠也将为其他对ROMK在非肾细胞（Gl tract， brain等）中的功能感兴趣的人提供资源。

DESCRIPTION (provided by applicant): ROMK (Kir1.1) forms apical K channels in the distal nephron and collecting duct that provide the K secretory pathways for 1) K recycling necessary for NaCI absorption by the thick ascending limb and 2) K secretion by connecting duct and principal cells. Therefore, defining the function and regulation of the ROMK channels is important for understanding renal K handling and body K homeostasis. Loss-of-function mutations in ROMK cause Bartter's syndrome, a hypotensive renal salt wasting disease, that is due to loss of salt reabsorbing capacity by the thick ascending limb. We have developed the only ROMK Bartter's mouse with sufficiently high survival (>30%) to permit physiological study - and is the only mouse model of Bartter's with such high survival. We propose to continue to study the pathophysiology of mouse ROMK Bartter's to enhance our understanding of the role of ROMK in renal K handling and to suggest potential modalities for therapy of human Bartter's syndrome. We will also develop two new ROMK transgenic mice that will provide models for assessing both the specific roles of ROMK isoforms and the regulated trafficking of ROMK isoforms in native kidney cells. One of these isoforms, ROMK1, is only expressed in late distal tubule and collecting duct and has been suggested to play an important role in the regulation of renal K secretion by dietary K intake. We propose to generate a mouse deficient only in ROMK1 by selective deletion of the ROMK1-specific exon using a Cre-LoxP strategy. We hypothesize that this mouse will not have a Bartter's phenotype, but instead, have disordered K handling with increases or decreases in dietary K intake. We will also generate a ROMK1 BAC-transgenic mouse tagged with FLAG and EGFP to define ROMK trafficking in kidney epithelial cells and to determine associated proteins in the ROMK regulatory complex. These mice will also provide a resource for others interested in ROMK function in non- renal cells (Gl tract, brain, etc).