Stoichiometry and modular retrieval of ENaC

上皮Na通道（ENaC）在建立血压中起着重要作用。ENaC活性是 部分是由其在质膜中的水平决定的。ENaC的膜水平反映了组成性递送， 规范检索ENaC中参与提取的机制和领域并不完全 明白我们感兴趣的是两个保守的，重叠的结构域（S/TPPPxYxS/TL）中发现的所有 ENaC亚基：PY（xPPxY）和基于酪氨酸的内吞（YxxL）基序。先前的基序靶向 通过Nedd 4泛素连接酶的泛素化通道;并且已知后者基序与mu 2相互作用 AP-2复合物的亚基，其靶向由发动蛋白介导的网格蛋白包被的小窝内吞作用的蛋白质。 我们将测试这些域是否是模块化的，因此是可分离的，或者它们是否一致行动。 生理信号级联（例如MAPK）通过促进通道降解来降低ENaC活性。 因此，我们还测试了MAPK信号通过这些模块化检索降低通道活性的假设。 域.此外，我们将确定是否绝对保守的S/T只是前面的PY和内 符合MAPK共有序列的YxxL基序是MAPK影响通道的靶点 活性和膜水平。ENaC是由3个不同亚基组成的异聚体通道， 含有两种或更少类型的活性降低的亚基。因此，亚单位的一个可能结果是， 回收是产生同源通道或仅含有两种类型亚基的通道。自从， 膜ENaC的亚基化学计量可能不是固定的，我们还问MAPK信号是否通过PY 和YxxL结构域改变ENaC亚基化学计量和/或组成以调节通道活性。 在这里，我们提出了四个具体的目标：1）确定膜ENaC的亚基化学计量; 2）确定 膜ENaC水平是否由模块化PY和YxxL内吞结构域控制， 3）确定生理细胞信号级联是否调节通道 通过PY和YxxL基序的修复以及保守S/T的差异磷酸化是否调节了这一点 4）确定ENaC亚基从质膜的回收是否是协调的。这 研究将提供关于ENaC的分子结构和这一重要的调控的重要见解。 频道

The epithelial Na channel (ENaC) plays a fundamental role in establishing blood pressure. ENaC activity is set, in part, by its level in the plasma membrane. Membrane levels of ENaC reflect constitutive delivery and regulated retrieval. The mechanisms and domains within ENaC involved in retrieval are not completely understood. We are interested in two conserved, overlying domains (S/TPPPxYxS/TL) found within all ENaC subunits: the PY (xPPxY) and tyrosine-based endocytic (YxxL) motifs. The prior motif targets the channel for ubiquitinylation via Nedd4 ubiquitin ligases; and the latter motif is known to interact with the mu2 subunit of the AP-2 complex, which targets proteins for clathrin coated-pit endocytosis mediated by dynamin. We will test whether these domains are modular and thus, separable or whether they act in concert. Physiological signaling cascades (e.g. MAPK) decrease ENaC activity by promoting channel degradation. Thus, we also test the hypothesis that MAPK signaling decreases channel activity via these modular retrieval domains. Moreover, we will determine whether absolutely conserved S/T just preceding the PY and within the YxxL motifs, which fit the consensus sequence for MAPK, are targets for MAPK impacting channel activity and membrane level. ENaC is a heteromeric channel comprised of 3 distinct subunits with channels containing two or fewer types of subunits having decreased activity. Thus, one possible outcome of subunit retrieval is production of homomeric channels or channels containing only two types of subunits. Since, subunit stoichiometry of membrane ENaC may not be fixed, we also ask whether MAPK signaling via the PY and YxxL domains changes ENaC subunit stoichiometry and/or composition to modulate channel activity. Here, we address four specific aims: 1) Determine subunit stoichiometry of membrane ENaC; 2) Determine whether membrane ENaC levels are controlled by modular PY and YxxL endocytic domains and assign significance to each domain; 3) Determine whether physiological cell signaling cascades modulate channel retrieval via the PY and YxxL motifs and whether differential phosphorylation of conserved S/T modulate this retrieval; and 4) Determine if retrieval of ENaC subunits from the plasma membrane is coordinated. This research will provide critical insight about the molecualr architecture of ENaC and regulation of this important channel.