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管细胞相对膜域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对目前尚不清楚的运输过程进行机制解释，该过程驱动极化定位，并从生理上调节集管中两个密切相关的通道（顶端分泌通道Kir1.1 （ROMK）和基侧膜通道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。具体而言，我们将解决以下关键和及时的问题：基底外侧膜Kir通道的极化运输信号是如何解释的？在这里，我们测试了一种新机制，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噬途径中的基底侧定向交通。2. 在钾适应过程中，Lin-7C相互作用是否调节肾皮质集管的基底外侧Kir通道？将对收集管特异性Lin-7C敲除小鼠进行研究，以验证钾适应中基底侧膜电导的增加是受Lin-7特异性异构体Lin-7C相互作用的影响。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，我们验证了Kir1.1中的“NPXY”型信号作为识别位点与一类新的网格蛋白接头蛋白ARH结合，这种相互作用标志着Kir1.1通道的快速内吞作用。此外，将使用ARH敲除小鼠来验证与ARH相互作用影响顶端膜活性Kir1.1通道生理衰减的假设。在这样做的过程中，调查计划将为支持健康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。

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.