Polarized Trafficking of K+ Channels 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的相对膜结构域上的极化表达确保了钾分泌过程的有效和精确控制，这对钾稳态至关重要。本提案的总体目标是开发一种机制解释的知之甚少的贩运过程，驱动极化定位和生理调节细胞表面密度的两个密切相关的渠道在集合管（顶端分泌通道，Kir1.1（ROMK），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，定义这些通道中的贩运信号，并阐明与它们相互作用的细胞内分选和保留机制。具体而言，我们将解决以下关键和及时的问题：1。基底外侧膜Kir通道中的极化运输信号如何解释？在这里，我们测试了一种新的机制，即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侧定向交通。2.在钾适应过程中，Lin-7 C相互作用是否调节肾皮质集合管基底外侧Kir通道？将检查集合管特异性Lin-7 C敲除小鼠以检验钾适应中基底外侧膜电导的增加受与特异性Lin-7同种型Lin-7 C的相互作用影响的假设。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，我们测试的假设，“NPXY”型信号Kir1.1作为识别位点结合到一个新的网格蛋白接头蛋白，ARH类的成员，这种相互作用标志着Kir1.1通道快速内吞作用。此外，ARH基因敲除小鼠将被用于测试的假设，Kir1.1通道在顶端膜活动的生理衰减的影响与ARH的相互作用。在这样做的过程中，调查计划将提供新的见解，以支持钾分泌的基本贩运机制，并了解当贩运信号和贩运机制在疾病中出错时会发生什么。 项目叙述：钾通道是钾平衡的基础，必须在肾脏的两个极化膜区域精确地组织起来，以实现有效的肾脏钾分泌。事实上，离子通道运输和表面表达的破坏可能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。在本提案中，我们阐明了在健康状态下驱动这些通道的膜运输的分子机制，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，这些研究应该提供新的见解，在健康和疾病的肾K处理和K稳态的分子基础，同时阐明膜蛋白靶向肾脏的基本机制。

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.