Molecular Basis of Potassium Channels in the Kidney

描述（由申请人提供）：本提案的总体目标是对远端肾元中驱动基底外侧膜定位和生理调节两个密切相关通道（Kir2.3和Kir4.1，与EAST/Sesame综合征相关的突变）的细胞表面密度的运输过程进行机制解释。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：基尔通道中的高尔基输出补丁如何影响基底外侧分选？与通常由短线性肽序列组成的传统运输信号不同，我们发现嵌入其三级结构的残基决定了典型钾基尔通道的高尔基出口。这个信号斑块形成了一个与AP1A接头复合体相互作用的识别位点，从而在反高尔基体上标记入网格蛋白包被囊泡的通道。在这里，我们验证了在Kir2.3和Kir4.1中发现的保守斑块信号通过选择通道作为包裹到网格蛋白包被囊泡中的货物来启动极化运输的假设。2. 如何解释基尔通道C端区域的基底侧贩运信号？根据我们发表的和初步的观察，我们提出一旦通道被标记为包含在网格蛋白包被的囊泡中，其他信号通过相互作用的基底外侧运输伴侣直接向基底外侧递送。3. 钾适应过程中肾皮质集管基底外侧Kir通道重构的基础是什么？该研究旨在验证一种假设，即以前未被认识到的Kir2.3和Kir4.1以及基底外侧PDZ保留复合物的Kir通道重塑过程，支持了钾适应中基底外侧膜电导的增加。通过解决这些问题，调查项目将为控制健康钾分泌的基本运输机制提供新的见解，并了解当运输信号和运输机制在疾病中出现问题时会发生什么。

DESCRIPTION (provided by applicant): The overarching goal of the present proposal is to develop a mechanistic explanation of the poorly understood trafficking processes that drive basolateral membrane localization and physiologically regulate the cell surface density of two closely related channels (Kir2.3 and Kir4.1, mutations in which are associated with EAST/Sesame syndrome) in the distal nephron. The program logically builds on our recent discoveries, defining the trafficking signals in these channels and the elucidating the intracellulr sorting and retention machinery that interact with them. Specifically, we will address the following critical and timely questions: 1. How does the Golgi Export patch in Kir channels influence basolateral sorting? Unlike conventional trafficking signals, which are typically comprised of short linear peptide sequences, we discovered that residues embedded its tertiary structure dictate Golgi exit of a prototypical potassium Kir channel. This signal patch forms a recognition site for interaction with the AP1A adaptor complex, thereby marking channels for incorporation into clathrin-coated vesicles at the trans- Golgi. Here we test the hypothesis that the conserved patch signal found in Kir2.3 and Kir4.1 initiates polarized trafficking by selecting channels as cargo for inclusion into clathrin-coated vesicles. 2. How are the basolateral trafficking signals in the C- terminal region of Kir channels interpreted? Based on our published and preliminary observation, we propose that once channels are marked for inclusion into clathrin-coated vesicles, other signals direct basolateral delivery by interacting basolateral trafficking chaperone(s). 3. What is the basis for basolateral Kir channel remodeling in the renal cortical collecting duct during potassium adaptation? This aim is designed to test the hypothesis that a previously unrecognized Kir channel remodeling process, involving Kir2.3 and Kir4.1 and a basolateral PDZ retention complex, underpins the increase in the basolateral membrane conductance in potassium adaptation. By addressing these questions, the program of investigation will provide new insights into the fundamental trafficking mechanisms that control potassium secretion in health and to understand what happens when trafficking signals and trafficking machiery goes wrong in disease.