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域上的极化表达确保了有效和精确地控制钾分泌过程，这对钾稳态至关重要。本提案的首要目标是从机制上解释目前知之甚少的运输过程，这些过程驱动极化定位，并在生理上调节收集管中两个密切相关的通道(顶端分泌通道Kir1.1(ROMK)和基底侧膜通道Kir2.3)的细胞表面密度。该计划在逻辑上建立在我们最近的发现基础上，定义了这些渠道中的贩运信号，并阐明了与它们相互作用的细胞内分选和保留机制。具体地说，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，我们测试了一种新的机制，通过该机制，Kir2.3中的独立信号与细胞内分选机制和PDZ支架复合体LIN-7/CASK顺序相互作用，以驱动生物合成和内吞途径中的基侧定向交通。2.在钾适应过程中，LIN-7C相互作用是否调节肾皮质集合管的基底外侧KIR通道？收集管特异性LIN-7C基因敲除小鼠将被检验，以检验钾适应中基侧膜电导增加受到特定LIN-7亚型LIN-7C相互作用的影响的假设。3.内吞作用控制Kir1.1(ROMK)通道密度的分子机制是什么？在这里，我们检验了一种假设，即Kir1.1中的“NPXY”型信号作为识别位点与一类新的笼蛋白适配器蛋白ARH结合，这种相互作用标志着Kir1.1通道具有快速内吞作用。此外，ARH基因敲除的小鼠将被用来检验这一假设，即Kir1.1通道在顶膜活动的生理性衰减受到与ARH的相互作用的影响。通过这样做，调查计划将为支持健康中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故障时会发生什么。 项目简介：支持钾平衡的钾通道必须精确地组织在肾脏的两个极化膜域，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): 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.