Polarized Trafficking of K+ Channels in the Kidney

超出所提供的空间。肾皮质集管（CCD）主细胞相对膜域上至少两种不同钾通道的极化运输、适当的表面表达和不同的调控确保了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）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层运输程序，包括一种新的生物合成分选过程和动态的PDZ依赖于基底外侧膜的保留。为了严格检验这一假设，将采用一种逐步多学科的方法，结合分子遗传学、细胞生物学、电生理学和转基因，来回答以下问题：在生物合成分选通路中如何解释Kit 2.3中的基底外侧运输信号？这一目的旨在严格测试新型生物合成分选机械候选物的作用。2. Kit 2.3的内化是否通过网格蛋白依赖机制发生，包括与AP2接头复合体的#亚基的直接相互作用？该目的旨在阐明参与Kit 2.3内吞运输的分子机制，为了解PDZ相互作用如何调节Kit 2.3表达提供背景。3. 是否通过限制内体运输与Lin-7/CASK PDZ复合物相互作用协调Kit 2.3的基底外侧表达。为了达到这个目的，我们将在缺乏和存在显性干扰性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？该目的旨在验证MOPP作为Lin 7 PDZ支架复合物的天然负调节因子的假设。5. 在钾适应过程中，Lin-7互作是否调控CCD中Kir 2.3的表达？在这个目的中，我们将确定Lin -7相互作用是否支持Kir 2.3的生理调节。将对野生型和Lin-7敲除小鼠进行研究。这些研究代表了主要研究者工作的及时和重要的延伸，并最终将为健康和疾病中肾脏钾处理和钾稳态的基础提供相当大的见解，同时阐明了控制肾脏膜蛋白分选的新的和目前尚未探索的机制。网站性能 ======================================== 节结束 ===========================================

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===========================================