Polarized Trafficking of K+ Channels in the Kidney

肾脏 K 通道的极化运输

• 批准号: 6835681
• 负责人: Paul A Welling
• 金额: $ 34.9万
• 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/6835681
• 关键词: Polarized; Trafficking; K+; Channels; Kidney

EXCEED THE SPACE PROVIDED. 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 # subnnit 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. PERFORMANCE SITE ========================================Section End===========================================

超出提供的空间。肾皮质集合管(CCD)主细胞上至少两个不同的钾通道的极化转运、适当的表面表达和不同的调节，确保了有效的钾分泌过程和钾的动态平衡。在这里，我们打算阐明控制基侧CCD通道KIR 2.3的极化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级转运计划，涉及一种新的生物合成分选过程和动态的、依赖于PDZ的基底外侧膜保留。为了严格验证这一假说，我们将采用分子遗传学、细胞生物学、电生理学和转基因相结合的逐步多学科方法来回答以下问题：1.如何解释Kit 2.3中的碱侧运输信号在生物合成分选途径中的作用？这一目标旨在批判性地测试新型生物合成分拣机械候选设备的作用。2.Kit 2.3的内化是否通过网状蛋白依赖的机制发生，涉及与AP2接头复合体的#subnnit直接相互作用。本研究旨在阐明Kit 2.3内吞转运的分子机制，为了解PDZ相互作用如何调控Kit 2.3的表达提供依据。3.与LIN-7/CAASK PDZ复合体相互作用，通过限制内体转运来协调Kit 2.3的基侧表达。在这一目标中，质膜周转率和外部标记通道的细胞内转运将在没有和存在显性干扰LIN-7结构的情况下进行评估。4.如何调节与独特的PDZ蛋白Mopp的相互作用来控制KIR 2.3的表面表达？这一目的是为了验证MOPP作为LIN 7 PDZ支架复合体的天然负调节因子的假设。5.在钾适应过程中，LIN-7的相互作用是否调节了CCDKIR 2.3的表达？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。野生型和LIN-7基因敲除小鼠将被研究。这些研究代表了首席研究员工作的及时和重要的扩展，并最终将为肾脏K处理和K动态平衡在健康和疾病中的基础提供相当大的洞察力，同时阐明控制肾脏膜蛋白分类的新的和目前未知的机制。表演网站========================================Section End===========================================