REGULATION OF ENAC BY INSERTION AND RETRIEVAL FROM CELL SURFACE

远端肾单位通过上皮细胞Na+吸收钠 ENaC通道是细胞外液的重要调节因子 容量和血压。删除细胞质的突变 β和Gamma ENaC亚基的c-teminus导致增加 钠离子吸收与高血压(利德尔综合征)。损失 与利德尔综合征相关的功能突变增加Na+ 通过增加等离子体中的通道数来增加电流 薄膜。猪瘟病毒C端序列PPPXYXXL突变 每个hEnaC亚基都重现了这些发现。有趣的是，这 序列类似于在许多 蛋白质。这个应用程序的目标是理解函数 和对hENaC的监管，以提供对基本机制的见解 钠离子转运和血压控制。我们提出三个建议 具体目的：1)探讨增重的机理(S) LIDELD~S突变引起的人ENaC的表面表达我们 将检验PPPXyXXL基序对 HENaC内化及其突变或缺失 Motif会降低渠道内部化的速度。我们还将 检验另一种假设，即利德尔~S突变会增加发病率 HENAC插入质膜的ATE。2)在 初步结果，我们发现hENaC的功能受 第二信使营地和库尔德工人党。我们将测试 假设这些第二信使调节hENaC功能 通过改变细胞表面的表达。我们还将测试备用的 他们改变频道评级的可能性。3)C-末端 HENaC亚基介导它们与其他细胞的相互作用 蛋白质。这样的相互作用很可能在 调控hENaC的表面表达和功能。在这 具体目标我们将确定与C- 末端PPPXyXXL基序。我们将测试特定的候选人 与hENaC相互作用的蛋白质。识别新的可疑对象 相互作用，我们还将筛选一个肾脏cDNA文库来鉴定 相互作用的蛋白质。在这一具体目标的第二部分，我们 将通过测试确定交互的功能重要性 它们对hENaC功能和表面表达的影响。这些 研究将帮助我们了解调节的基本机制 HENAC和Na+吸收，并可能为 高血压的分子机制。

Sodium absorption in the distal nephron through the epithelial Na+ channel, ENaC, is an important regulator of extracellular fluid volume and blood pressure. Mutations that delete the cytoplasmic c-teminus of the Beta and Gamma ENaC subunits cause increase Na+ absorption and hypertension (Liddle's syndrome). Loss of function mutations associated with Liddle's syndrome increase Na+ current by increasing the number of channels in the plasma membrane. Mutation of the C-terminal sequence PPPXYXXL in each hEnaC subunit reproduced these findings. Interestingly, this sequence is similar to internalization motifs found in a number of proteins. The goal of this application is to understand the function and regulation of hENaC to provide insight into basic mechanisms of Na+ transport and blood pressure control. we propose three specific aims; 1) To investigate the mechanism(s) of increased surface expression of hENaC caused by Liddle~s mutations. We will test the hypothesis that the PPPXyXXL motif is important for the internalization of hENaC, and that mutation or deletion of this motif decreases the rate of channel internalization. We will also test the alternate hypothesis that Liddle~s mutations increase the rate ate of insertion of hENAC into the plasma membrane. 2) In preliminary results, we found that hENaC function is regulated by the second-messengers cAMP and PKC. We will test the hypothesis that these second-messengers regulate hENaC function by altering cell surface expression. We will also test the alternate possibility that they alter channel rating. 3) The C-terminus of hENaC subunits mediate their interaction with other cellular proteins. It is likely that such interactions are important in controlling surface expression and function of hENaC. In this Specific Aim we will identify proteins that interact with the C- terminal PPPXyXXL motif. We will test specific candidate proteins for interaction with hENaC. To identify new unsuspected interactions, we will also screen a kidney cDNA library to identify interacting proteins. In the second part of this Specific Aim, we will determine the functional significance of interactions by testing their affect on hENaC function and surface expression. These studies will help us learn about basic mechanisms of regulation of hENAC and Na+ absorption, and may provide new insights into molecular mechanisms of hypertension.