Polarized Trafficking of K+ Channels in the Kidney

肾脏 K 通道的极化运输

• 批准号: 7568916
• 负责人: Paul A Welling
• 金额: $ 31.88万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7568916
• 关键词: Adaptor Signaling Protein; Address; Apical; Arts; Automobile Driving; Binding Sites; Biological; Cell membrane; Cell surface; Cells; Cellular biology; Clathrin Adaptors; Complex; Data; Dietary Potassium; Disease; Duct (organ) structure; Electrophysiology (science); Endocytosis; Equilibrium; Goals; Health; Homeostasis; Imaging Techniques; Investigation; Ion Channel; KCNJ1 gene; KCNJ1 protein; Kidney; Knockout Mice; Location; Membrane; Membrane Protein Traffic; Membrane Proteins; Methods; Minerals; Molecular; Molecular Genetics; PTB Domain; Pathway interactions; Physiological; Physiology; Potassium; Potassium Channel; Principal Investigator; Process; Protein Isoforms; Publishing; Retrieval; Route; Signal Transduction; Sodium Chloride; Sorting - Cell Movement; Surface; Testing; Work; apical membrane; attenuation; base; basolateral membrane; density; design; insight; interdisciplinary approach; member; membrane activity; novel; programs; protein transport; prototype; scaffold; secretion process; src-Family Kinases; trafficking

DESCRIPTION (provided by applicant): Polarized expression of different potassium channel subtypes on opposite membrane domains of renal collecting duct cells insures an efficient and precisely controlled potassium secretion process, critical for potassium homeostasis. The overarching goal of the present proposal is to develop a mechanistic explanation of the poorly understood trafficking processes that drive polarized localization and physiologically regulate the cell surface density of two closely related channels in the collecting duct (the apical secretory channel, Kir1.1 (ROMK), and a basolateral membrane channel, Kir2.3). The program logically builds on our recent discoveries, defining the trafficking signals in these channels and the elucidating the intracellular sorting and retention machinery that interacts with them. Specifically, we will address the following critical and timely questions: 1. How are the polarized trafficking signals in basolateral membrane Kir channels interpreted? Here, we test a novel mechanism whereby independent signals in Kir2.3 sequentially interact with intracellular sorting machinery and a PDZ scaffold complex, Lin-7/CASK, to drive basolateral-directed traffic in the biosynthetic and endocytotic pathways. 2. Does Lin-7C interaction regulate basolateral Kir channel in the renal cortical collecting duct during potassium adaptation? Collecting duct specific Lin-7C knockout mice will be examined to test the hypothesis that the increase in basolateral membrane conductance in potassium adaptation is influenced by interaction with a specific Lin-7 isoform, Lin-7C. 3. What is the molecular mechanism by which Kir1.1 (ROMK) channel density is controlled by endocytosis. Here we test the hypothesis that a "NPXY"-type signal in Kir1.1 serves as recognition site for binding to a member of a new class of clathrin-adaptor proteins, ARH, and this interaction marks Kir1.1 channels for rapid endocytosis. Moreover, ARH knockout mice will be used to test the hypothesis that physiological attenuation of Kir1.1 channels at the apical membrane activity is influenced by interaction with ARH. In doing so, the program of investigation will provide new insights into the fundamental trafficking mechanisms that underpin potassium secretion in health and to understand what happens when trafficking signals and trafficking machinery goes wrong in disease. Project Narrative: Potassium channels that underpin potassium balance must be precisely organized at two polarized membrane domains in the Kidney for efficient renal potassium secretion. Disruption of ion 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.

描述（由申请人提供）：不同钾通道亚型在肾集合管细胞相对膜域上的极化表达确保了有效且精确控制的钾分泌过程，这对于钾稳态至关重要。本提案的总体目标是对人们知之甚少的运输过程进行机械解释，这些过程驱动极化定位并在生理上调节集合管中两个密切相关的通道（顶端分泌通道，Kir1.1（ROMK）的细胞表面密度）和基底外侧膜通道，Kir2.3）。该计划在逻辑上建立在我们最近的发现之上，定义了这些通道中的运输信号，并阐明了与它们相互作用的细胞内分选和保留机制。具体来说，我们将解决以下关键和及时的问题：1.如何解释基底外侧膜Kir通道中的极化运输信号？在这里，我们测试了一种新机制，Kir2.3 中的独立信号依次与细胞内分选机制和 PDZ 支架复合物 Lin-7/CASK 相互作用，以驱动生物合成和内吞途径中的基底外侧定向交通。 2. 在钾适应过程中，Lin-7C 相互作用是否调节肾皮质集合管中的基底外侧 Kir 通道？将检查集合管特异性 Lin-7C 敲除小鼠，以测试钾适应中基底外侧膜电导的增加受到与特定 Lin-7 亚型 Lin-7C 相互作用的影响的假设。 3. Kir1.1（ROMK）通道密度受内吞作用控制的分子机制是什么？在这里，我们测试了这样的假设：Kir1.1 中的“NPXY”型信号充当与新型网格蛋白适配器蛋白 ARH 的成员结合的识别位点，并且这种相互作用标记了 Kir1.1 快速内吞作用的通道。此外，ARH 敲除小鼠将用于测试顶膜活性 Kir1.1 通道的生理衰减受到与 ARH 相互作用的影响的假设。在此过程中，该调查计划将为健康中支撑钾分泌的基本贩运机制提供新的见解，并了解当贩运信号和贩运机制在疾病中出现问题时会发生什么。 项目叙述：支持钾平衡的钾通道必须在肾脏的两个极化膜域上精确组织，以实现肾脏有效的钾分泌。事实上，离子通道运输和表面表达的破坏会对盐和矿物质平衡产生破坏性后果。尽管其重要性，细胞生物学和生理学中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。在本提案中，我们阐明了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会发生什么。因此，这些研究应该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，同时阐明肾脏中膜蛋白靶向的基本机制。