ENAC FUNCTION, REGULATION, AND ION PERMEATION

描述（改编自申请人的摘要）： 远端肾单位通过上皮Na+通道，ENaC，是重要的 细胞外液量和血压的调节器。的突变 删除β和γ hENaC亚基的细胞质C端， Na+吸收增加和高血压（Liddle综合征）。损失 hENaC功能突变导致Na+消耗（假性醛固酮减少症型 1）。在初步研究中，与利德尔综合征相关的突变 通过增加血浆中通道的数量来增加Na+电流 膜的每个hENaC中C末端序列PPPXYXXL的突变 亚组再现了这些发现。有趣的是，这个序列 在许多蛋白质中发现的内化基序。这个目标 应用是为了了解hENaC的功能和调节， 深入了解Na+转运和血压控制的基本机制。 具体目的有三个：1）研究 由Liddle突变引起的hENaC的表面表达增加。的 要检验的假设是PPPXYXXL基序对于 hENaC内化，以及该基序的突变或缺失， 降低了通道内化的速率。另一种假设， Liddle的突变增加了hENaC插入细胞的速率， 2）初步结果表明，hENaC在细胞膜上的表达与细胞膜的表达有关， 其功能由第二信使cAMP和PKC调节。的假设 这些第二信使通过以下方式调节hENaC功能： 改变细胞表面表达。另一种假设是，它们改变了 通道门控，也将被测试;和3）ENaC的一个重要属性是 其对Na+的选择性高于对K+的选择性。这是由氨基决定的 所述残基衬在通道孔中。这三个小组中 有助于孔将被确定和特定的残留物，线 通过半胱氨酸残基的共价修饰来鉴定孔。 他们的初步结果发现，在第二个半胱氨酸， γ ENaC的跨膜段排列在通道孔中。使用 定点诱变，他们将确定是否类似的残基， α和β亚基也排列在孔中。他们也会利用这个 鉴定ENaC中衬孔的其他残基的策略， 开始定义孔的结构，及其分子基础， 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.