Structure and Function of ROMK Channel in Kidney

描述(申请人提供)：ROMK(Kir1.1)在远端肾单位和集合管形成顶端K通道，为1)粗大的升肢吸收NaCI所需的K循环和2)连接管和主细胞的K分泌提供通道。因此，明确ROMK通道的功能和调节对于了解肾脏K的处理和体内K的动态平衡是非常重要的。ROMK功能缺失突变导致巴特综合征，这是一种低血压的肾性盐耗疾病，是由于粗大的上升肢失去盐分再吸收能力所致。我们已经培育出了唯一的ROMK巴特小鼠，存活率足够高(30%)，可以进行生理研究--也是巴特小鼠中唯一存活率如此高的小鼠模型。我们建议继续研究小鼠ROMK巴特综合征的病理生理学，以加强我们对ROMK在肾脏K处理中的作用的理解，并为人类巴特综合征的治疗提供潜在的方法。我们还将开发两种新的ROMK转基因小鼠，它们将为评估ROMK亚型的特定作用和ROMK亚型在天然肾脏细胞中的调节运输提供模型。其中一个亚型ROMK1仅在远曲小管和集合管的晚期表达，并被认为在饮食K摄入量调节肾脏K的分泌中起重要作用。我们建议通过使用CRE-loxP策略选择性地删除ROMK1特异的外显子来产生一只ROMK1缺陷的小鼠。我们推测，这只小鼠不会有巴特氏表型，而是随着饮食中钾摄入量的增加或减少而出现钾处理紊乱。我们还将产生一只标记有FLAG和EGFP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质。这些小鼠也将为其他对ROMK在非肾细胞(Gl道、脑等)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).