Polarized Trafficking of K+ Channels in the Kidney

肾脏 K 通道的极化运输

• 批准号: 7000368
• 负责人: Paul A Welling
• 金额: $ 34.08万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7000368
• 关键词: Polarized; Trafficking; K+; Channels; Kidney

DESCRIPTION (provided by applicant): Polarized trafficking, appropriate surface expression and disparate regulation of at least two different potassium channels on opposite membrane domains of the renal cortical collecting duct (CCD) principal cell insure an efficient potassium secretion process and potassium homeostasis. Here, we propose to elucidate the molecular mechanisms governing polarized targeting and surface expression of the basolateral CCD channel, Kir 2.3. Our previous work suggests a hierarchical trafficking program, involving a novel biosynthetic sorting process and dynamic, PDZ dependent retention at the basolateral membrane. To critically test this hypothesis, a stepwise multidisciplinary approach, combining molecular genetics, cellular biology, electrophysiology and transgenics, will be employed to answer the following questions: 1. How is the basolateral trafficking signal in Kit 2.3 interpreted within the biosynthetic sorting pathway? This aim is designed to critically test the role of novel biosynthetic sorting machinery candidates. 2. Does internalization of Kit 2.3 occur via clathrin-dependent mechanism, involving a direct interaction with the ? subunit of AP2 adaptor complex. This aim is designed to elucidate the molecular mechanisms involved in endocytotic trafficking of Kit 2.3, providing a context to understand how PDZ interactions regulate Kit 2.3 expression. 3. Does interaction with the Lin-7/CASK PDZ complex coordinate basolateral expression of Kit 2.3 by limiting endosomal trafficking. In this aim, plasma membrane turnover rate and intracellular trafficking of externally tagged channels will be assessed in the absence and presence of dominant interfering Lin-7 constructs. 4. How is interaction with MOPP, a unique PDZ protein, regulated to control surface expression of Kir 2.3? This aim is designed to test the hypothesis that MOPP acts as a natural negative regulator of Lin 7 PDZ scaffolding-complexes. 5. Does Lin-7 interaction regulate Kir 2.3 expression in the CCD during potassium adaptation? In this aim, we will determine if Lin -7 interaction underpins the physiological regulation of Kir 2.3. Wild-type and Lin-7 knockout mice will be studied. These studies represent a timely and important extension of the principal investigator's work, and should ultimately provide considerable insight into the basis of renal K handling and K homeostasis in health and disease while illuminating new and presently unexplored mechanisms controlling membrane-protein sorting in the kidney.

描述（由申请人提供）：肾皮质集合管（CCD）主细胞相对膜域上至少两个不同钾通道的极化运输、适当的表面表达和不同的调节确保有效的钾分泌过程和钾稳态。在这里，我们建议阐明控制基底外侧 CCD 通道 Kir 2.3 的偏振靶向和表面表达的分子机制。我们之前的工作提出了一种分层运输程序，涉及一种新颖的生物合成分选过程和动态的、PDZ 依赖性的基底外侧膜保留。为了严格检验这一假设，将采用逐步的多学科方法，结合分子遗传学、细胞生物学、电生理学和转基因学来回答以下问题： 1. 如何在生物合成分选途径中解释试剂盒 2.3 中的基底外侧运输信号？这一目标旨在严格测试新型生物合成分选机器候选者的作用。 2. Kit 2.3 的内化是否通过网格蛋白依赖性机制发生，涉及与 的直接相互作用？ AP2 接头复合体的亚基。该目的旨在阐明 Kit 2.3 内吞运输所涉及的分子机制，为理解 PDZ 相互作用如何调节 Kit 2.3 表达提供背景。 3. 与 Lin-7/CASK PDZ 复合物的相互作用是否通过限制内体运输来协调 Kit 2.3 的基底外侧表达。在此目的中，将在不存在和存在显性干扰 Lin-7 构建体的情况下评估质膜周转率和外部标记通道的细胞内运输。 4. 如何调节与 MOPP（一种独特的 PDZ 蛋白）的相互作用来控制 Kir 2.3 的表面表达？该目的旨在检验 MOPP 作为 Lin 7 PDZ 支架复合物的天然负调节剂的假设。 5. 在钾适应过程中，Lin-7 相互作用是否调节 CCD 中的 Kir 2.3 表达？为此，我们将确定 Lin -7 相互作用是否支持 Kir 2.3 的生理调节。将研究野生型和 Lin-7 基因敲除小鼠。这些研究代表了主要研究者工作的及时而重要的扩展，并最终应为健康和疾病中肾脏钾处理和钾稳态的基础提供相当多的见解，同时阐明控制肾脏中膜蛋白分选的新的和目前尚未探索的机制。