Stoichiometry and modular retrieval of ENaC

描述（由申请人提供）：上皮钠通道（ENaC）在血压的建立中起着重要作用。ENaC活性部分取决于其在质膜中的水平。ENaC的膜水平反映了本构传递和调控回收。ENaC中涉及检索的机制和领域尚未完全了解。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。先前的基序通过Nedd4泛素连接酶靶向泛素化通道；后一个基序已知与AP-2复合物的mu2亚基相互作用，AP-2复合物的靶蛋白是由动力蛋白介导的网格蛋白包覆核内吞作用。我们将测试这些领域是否模块化，因此，可分离的，或者他们是否一致行动。生理信号级联（如MAPK）通过促进通道降解降低ENaC活性。因此，我们也验证了MAPK信号通过这些模块化检索域降低通道活性的假设。此外，我们将确定在PY之前和YxxL基序内的绝对保守S/T是否符合MAPK的共识序列，是MAPK影响通道活性和膜水平的目标。ENaC是由3个不同的亚基组成的异质通道，其中含有两种或更少类型的亚基的通道活性降低。因此，亚基检索的一个可能结果是产生同质通道或仅包含两种亚基的通道。由于ENaC膜的亚基化学计量可能不是固定的，我们也想知道MAPK信号是否通过PY和YxxL结构域改变ENaC亚基化学计量和/或组成来调节通道活性。在这里，我们有四个具体的目标：1)确定膜ENaC的亚基化学计量；2)确定膜ENaC水平是否受模块化的PY和YxxL内吞结构域控制，并对每个结构域进行显著性划分；3)确定生理细胞信号级联是否通过PY和YxxL基序调节通道恢复，以及保守S/T的差异磷酸化是否调节这种恢复；4)确定从质膜中提取ENaC亚基是否协调一致。这项研究将为ENaC的分子结构和这一重要通道的调控提供关键的见解。

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.