Molecular Mechanism of ROMK Channel Function

描述（由申请人提供）：肾脏中的ROMK （Kir 1.1， KCNJ1基因产物）通道被精细调控，调节肾脏钾排泄，维持钾平衡。网格蛋白依赖的胞吞作用在钾缺乏症中起关键作用，限制尿钾流失。在肾脏疾病中，异常的ROMK内吞可能导致钾潴留和危及生命的高钾血症。现有证据表明，在一种常见的高钾血症和高血压疾病中发生突变的激酶WNKs会刺激ROMK内吞。这一应用建立在我们发现ROMK中“NPXY”型信号的新变体作为ARH结合的识别位点，ARH是一类新的网格蛋白衔接蛋白的成员，这种相互作用标志着快速内吞和最终溶酶体降解的通道。缺乏ARH的敲除小鼠对饮食中钾的摄入表现出肾脏ROMK的改变。为了将这些突破性的观察结果带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，以批判性地评估ARH依赖性ROMK内吞的生理后果；2)探索一种新的信号通路对ARH进行生理调节；3)阐明WNK-1刺激ARH依赖性内吞和ROMK向溶酶体的内吞后通路的分子机制。这些研究将为健康和疾病中肾脏钾处理和钾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. PUBLIC HEALTH RELEVANCE: ROMK potassium channels are tightly regulated in the kidney by membrane trafficking mechanisms, ensuring that potassium is precisely excreted in accord with the demands of potassium balance. Disruption of ROMK channel trafficking and surface expression can, in fact, have devastating consequences on salt and mineral balance. Despite its importance, a long-standing and fundamental question in cell biology and physiology has been how the number and location of these membrane proteins are precisely controlled. In the present proposal, we elucidate the molecular mechanisms driving membrane trafficking of these channels in health and study what may happen when these processes go awry in disease. Thus, 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.