Molecular Mechanism of ROMK Channel Function

描述(申请人提供)：肾脏中的ROMK(KIR 1.1，他的KCNJ1基因产物)通道被精细地调节，以调节肾脏的钾排泄，维持钾平衡。依赖于笼蛋白的内吞作用在钾缺乏时起着关键作用，限制了尿钾的丢失。在肾脏疾病中，异常的ROMK内吞作用可能导致钾滞留和危及生命的高钾血症。现有证据表明，ROMK内吞作用是由WNKS刺激的，WNKS是一种常见的高钾血症和高血压疾病中突变的激酶。这一应用建立在我们的发现基础上，即ROMK中的一个“NPXY”型信号的新变体作为与ARH结合的识别位点，ARH是一类新的笼蛋白适配器蛋白的成员，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的基因敲除小鼠对饮食中钾的摄入表现出肾脏ROMK反应的改变。为了使这些突破性的观察结果对钾平衡的实现有一个全新的理解，我们概述了以下计划：1)对ARH基因敲除小鼠进行完整的系统到分子表型鉴定，以关键地评估ARH依赖的ROMK内吞作用的生理后果；2)探索生理调节ARH的新信号通路的参与；3)阐明WNK-1刺激ARH依赖的内吞作用和ROMK内吞后路由到溶酶体的分子机制。这些研究将为肾脏K处理和K动态平衡在健康和疾病中的分子基础提供新的见解，同时阐明肾脏中膜蛋白靶向的基本机制。 与公众健康相关：ROMK钾通道在肾脏中通过膜转运机制受到严格调控，确保钾准确地排出，符合钾平衡的要求。事实上，ROMK通道贩运和表面表达的中断可能会对盐分和矿物质平衡造成毁灭性的后果。尽管它很重要，但在细胞生物学和生理学中，一个长期存在的基本问题是如何精确控制这些膜蛋白的数量和位置。在目前的提案中，我们阐明了在健康中驱动这些通道膜运输的分子机制，并研究了当这些过程在疾病中出错时可能发生的情况。因此，这些研究将为肾脏K处理和K动态平衡在健康和疾病中的分子基础提供新的见解，同时阐明肾脏中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.