Stoichiometry and modular retrieval of ENaC

描述(由申请人提供)：上皮钠通道(ENaC)在建立血压中起着基础性作用。ENAC活性在一定程度上是由其在质膜中的水平决定的。ENaC的膜水平反映了结构性递送和调节的检索。ENaC内涉及检索的机制和结构域尚未完全了解。我们感兴趣的是在所有ENaC亚基中发现的两个保守的覆盖结构域(S/TPPPxYxS/TL)：PY(XPPxY)和基于酪氨酸的内吞(YxxL)基序。前一个基序通过Nedd4泛素连接酶靶向泛素化通道；后一个基序与AP-2复合体的MU2亚基相互作用，后者靶向由Dynamin介导的笼蛋白包裹的凹坑内吞作用的蛋白质。我们将测试这些域是否是模块化的，从而是可分离的，还是它们是否协同工作。生理信号级联(如MAPK)通过促进通道降解来降低ENaC的活性。因此，我们还验证了MAPK信号通过这些模块检索结构域减少通道活动的假设。此外，我们将确定PY之前和YxxL基序中的绝对保守的S/T是否是影响通道活性和膜水平的MAPK的靶标。ENAC是由3个不同亚基组成的异构体通道，通道包含两种或更少类型的活性降低的亚基。因此，亚基检索的一个可能的结果是产生同源通道或仅包含两种类型的亚基的通道。由于膜ENaC的亚单位化学计量可能不是固定的，我们还询问通过PY和YxxL结构域的MAPK信号是否改变了ENaC亚单位的化学计量和/或组成来调节通道活性。在这里，我们针对四个具体目标：1)确定膜ENaC的亚单位化学计量；2)确定膜ENaC的水平是否受模块化的PY和YxxL内含域的控制，并赋予每个结构域意义；3)确定生理细胞信号级联是否通过PY和YxxL基序调节通道检索，以及保守的S/T的差异磷酸化是否调节这种检索；以及4)确定从质膜中ENaC亚单位的检索是否协调。这项研究将对ENaC的分子结构和这一重要通道的调控提供重要的见解。

DESCRIPTION (provided by applicant): 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 molecular architecture of ENaC and regulation of this important channel.