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人们知之甚少的运输过程进行机械解释，这些运输过程驱动基底外侧膜定位并在生理上调节远端肾单位中两个密切相关的通道（Kir2.3和Kir4.1，其中的突变与EAST/Sesame综合征相关）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。具体来说，我们将解决以下关键且及时的问题： 1. Kir通道中的高尔基体输出斑块如何影响基底外侧排序？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高尔基体离开原型钾Kir通道。该信号贴片形成与 AP1A 接头复合物相互作用的识别位点，从而标记掺入反高尔基体处网格蛋白包被的囊泡的通道。在这里，我们测试了这样的假设：Kir2.3 和 Kir4.1 中发现的保守斑块信号通过选择通道作为货物包含到网格蛋白包被的囊泡中来启动极化运输。 2.如何解释Kir通道C端区域的基底外侧运输信号？根据我们发表的初步观察，我们建议一旦通道被标记为包含在网格蛋白包被的囊泡中，其他信号就会通过相互作用的基底外侧运输伴侣来指导基底外侧递送。 3.钾适应过程中肾皮质集合管基底外侧Kir通道重塑的基础是什么？该目的旨在检验以下假设：先前未被识别的 Kir 通道重塑过程（涉及 Kir2.3 和 Kir4.1 以及基底外侧 PDZ 保留复合体）支持钾适应中基底外侧膜电导的增加。通过解决这些问题，该调查计划将为控制健康中钾分泌的基本贩运机制提供新的见解，并了解当疾病中的贩运信号和贩运机制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.