ENAC FUNCTION, REGULATION, AND ION PERMEATION

描述(改编自申请人的摘要)：钠在 远端肾单位通过上皮性Na+通道，ENaC是一个重要的 细胞外液容量和血压的调节器。突变 删除导致β和γhENaC亚基的细胞质C-teminus 钠离子吸收增加和高血压(利德尔综合征)。损失 HENaC功能突变导致Na+消耗(假性低醛固酮增多症 1)。在初步研究中，与利德尔综合征相关的突变 通过增加血浆中的通道数来增加钠电流 薄膜。各hENaC中C-末端序列PPPXYXXL的突变 亚基重现了这些发现。有趣的是，这个序列是相似的 一些蛋白质中发现的内化基序。这样做的目的是 应用是为了了解hENaC的功能和调节，为提供 深入了解钠离子转运和血压控制的基本机制。 具体有三个目的：1)探讨其发病机制(S) 利德尔突变引起的hENaC表面表达增加。这个 有待检验的假设是，PPPXYXXL基序对 HENaC内化，以及该基序的突变或缺失， 降低渠道内部化的速度。另一种假设是， 利德尔的突变增加了hENaC插入到 质膜，也将进行测试；2)初步结果显示，hENaC 其功能受第二信使cAMP和PKC的调节。假说 有待检验的是，这些第二信使通过以下方式调节hENaC功能 改变细胞表面的表达。另一种假设是，它们改变了 3)ENaC的一个重要特性是 它对Na+的选择性高于对K+的选择性。这是由氨基决定的 渠道毛孔内的助剂残留物。这三个亚基中的哪一个 毛孔的贡献将被确定和具体的残留物线条 将通过半胱氨酸残基的共价修饰来识别孔洞。 他们的初步结果发现，第二种中的两种半胱氨酸 伽马ENaC的跨膜段排列在通道孔内。vbl.使用 定点突变，他们将确定类似的残留物是否在 α和β亚基也排列在毛孔中。他们也会用这个 确定ENaC中排列在孔隙中的其他残留物的策略，以便 开始定义毛孔的结构，以及它的分子基础 离子选择性。

DESCRIPTION (Adapted from the Applicant's Abstract): 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 hENaC subunits cause increased Na+ absorption and hypertension (Liddle's syndrome). Loss of function mutations in hENaC cause Na+ wasting (pseudohypoaldosteronism type 1). In preliminary studies, 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. There are three Specific Aims: 1) To investigate the mechanism(s) of increased surface expression of hENaC caused by Liddle's mutations. The hypothesis to be tested is 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. An alternate hypothesis, that Liddle's mutations increase the rate of insertion of hENaC into the plasma membrane, will also be tested; 2) Preliminary results show that hENaC function is regulated by the second-messengers cAMP and PKC. The hypothesis to be tested is that these second-messengers regulate hENaC function by altering cell surface expression. An alternate hypothesis, that they alter channel gating, will also be tested; and 3) An important property of ENaC is its high degree of selectivity for Na+ over K+. This is determined by amino aid residues that line the channel pore. Which of the three subunits contribute to the pore will be determined and specific residues that line the pore will be identified, by covalent modification of cysteine residues. Their preliminary results found that two cysteines in the second membrane-spanning segment of gamma ENaC line the channel pore. Using site-directed mutagenesis, they will determine whether analogous residues in the alpha and beta subunits also line the pore. They will also use this strategy to identify other residues in ENaC that line the pore in order to begin to define the structure of the pore, and the molecular basis of its ion selectivity.