Molecular Basis of Potassium Channels in the Kidney

描述(由申请人提供)：本提案的主要目标是从机制上解释鲜为人知的转运过程，这些过程驱动基底膜定位和生理调节远端肾单位两个密切相关通道(Kir2.3和Kir4.1，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拣和保留机制。具体地说，我们将解决以下关键和及时的问题：1.KIR渠道中的高尔基出口补丁如何影响基线分类？与通常由短线性多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的KIR通道的高尔基退出。这个信号补丁形成一个识别位点，与AP1a适配器复合体相互作用，从而标记通道，以便在反式高尔基体上整合到笼蛋白包裹的小泡中。在这里，我们测试了在Kir2.3和Kir4.1中发现的保守的斑块信号通过选择通道作为货物进入笼蛋白包裹的小泡而启动极化运输的假设。2.如何解释KIR航道C-终端区域的基侧贩运信号？基于我们已发表的和初步的观察，我们认为一旦通道被标记为包含在笼蛋白包裹的小泡中，其他信号就会通过相互作用的基侧运输伴侣蛋白(S)直接基侧输送。3.钾适应过程中肾皮质集合管基底外侧KIR通道重构的基础是什么？这一目的是为了验证一种假说，即先前未知的KIR通道重塑过程，包括Kir2.3和Kir4.1以及基侧PDZ保留复合体，支持了钾适应中基侧膜电导的增加。通过解决这些问题，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): 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.