REGULATION OF ENAC BY INSERTION AND RETRIEVAL FROM CELL SURFACE

远端肾单位通过上皮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γ ENaC亚基的C末端导致 Na+吸收和高血压（Liddle综合征）。 损失 与Liddle综合征相关的功能突变增加Na+ 通过增加等离子体中通道的数量来增加电流 膜的 突变的C-末端序列PPPXYXXL， 每个hEnaC亚基再现了这些发现。 有趣的是， 序列类似于在许多细胞中发现的内化基序。 proteins. 此应用程序的目标是了解函数 和hENaC的调节，以深入了解基本机制 钠离子转运和血压控制。 我们提出了三 具体目标; 1）调查增加的机制 由Liddle~s突变引起的hENaC的表面表达。 我们 将检验PPPXyXXL基序对以下假设的重要性： hENaC内在化，以及这种内在化的突变或缺失 基序降低通道内化的速率。 我们还将 检验Liddle~s突变增加 hENAC插入质膜的速率。 2)在 初步结果，我们发现hENaC的功能是由 第二信使cAMP和PKC。 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 改变频道评级的可能性。 3)的c末端 hENaC亚基介导它们与其它细胞因子的相互作用。 proteins. 这种相互作用很可能在以下方面很重要： 控制hENaC的表面表达和功能。 在这 具体目标我们将确定蛋白质的相互作用与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。 找出新的未被怀疑的 相互作用，我们还将筛选肾脏cDNA文库， 相互作用的蛋白质 在第二部分的具体目标，我们 将通过测试确定相互作用的功能意义 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基本的调节机制， hENAC和Na+吸收，并可能提供新的见解， 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.