Molecular Mechanism of ROMK Channel Function

描述（申请人提供）：肾脏中的ROMK（Kir 1.1，KCNJ 1基因的产物）通道被精细调节以调节肾脏钾排泄并维持钾平衡。网格蛋白依赖的内吞作用起着关键作用，限制钾缺乏时的尿钾损失。在肾脏疾病中，异常的ROMK内吞作用可能导致钾潴留和危及生命的高钾血症。现有的证据表明ROMK内吞作用是由WNK刺激的，WNK是在高钾血症和高血压的常见疾病中突变的激酶。这个应用程序建立在我们的发现，一个新的变体的“NPXY”型信号在ROMK作为识别位点结合ARH，一个新的网格蛋白接头蛋白类的成员，这种相互作用标志着通道快速内吞作用和最终的溶酶体降解。缺乏ARH的基因敲除小鼠表现出对膳食钾摄入的肾脏ROMK反应的改变。为了将这些突破性的观察结果推向对钾平衡如何实现的全新理解，我们概述了以下计划：1）对ARH敲除小鼠进行完整的系统-分子表型表征，以严格评估ARH依赖性ROMK内吞作用的生理学后果，2）探索生理学调节ARH的新信号传导途径的参与，3）阐明WNK-1刺激ARH依赖的内吞作用和ROMK内吞后向溶酶体的途径的分子机制。这些研究将为健康和疾病中肾脏K处理和K稳态的分子基础提供新的见解，同时阐明肾脏膜蛋白靶向的基本机制。

DESCRIPTION (provided by applicant): ROMK (Kir 1.1, product of he KCNJ1 gene) channels in the kidney are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and life-threatening hyperkalemia. Available evidence indicates ROMK endocytosis is stimulated by WNKs, kinases that are mutated in a familiar disease of hyperkalemia and hypertension. This application builds on our discoveries that a novel variant of a "NPXY"-type signal in ROMK serves as a recognition site for binding to ARH, a member of a new class of clathrin-adaptor proteins, and this interaction marks channels for rapid endocytosis and eventual lysosomal degradation. Knockout mice, lacking ARH, exhibit an altered renal ROMK response to dietary potassium intake. To carry these breakthrough observations toward a completely new understanding of how potassium balance is achieved, we outline plans to: 1) conduct a complete system-to-molecule phenotypic characterization of the ARH knockout mouse to critically evaluate the physiological consequence of ARH-dependent ROMK endocytosis, 2) explore the involvement of a novel signaling pathway that physiologically regulates ARH, 3) elucidate the molecular mechanism by which WNK-1 stimulates ARH-dependent endocytosis and post-endocytic routing of ROMK to the lysosome. The studies should provide novel insights into the molecular basis of renal K handling and K homeostasis in health and disease while illuminating fundamental mechanisms of membrane protein targeting in the kidney.