Stoichiometry and modular retrieval of ENaC

ENaC 的化学计量和模块化检索

• 批准号: 7088155
• 负责人: James D Stockand
• 金额: $ 28.04万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088155
• 关键词: biological signal transduction; cell membrane; endocytosis; fluorescence microscopy; fluorescence resonance energy transfer; mitogen activated protein kinase; protein degradation; protein kinase C; protein protein interaction; protein structure function; sodium channel; stoichiometry

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.

描述（由申请人提供）：上皮NA通道（ENAC）在建立血压中起着基本作用。 ENAC活性在质膜中部分设置为其水平。 ENAC的膜水平反映了本构的递送和调节的检索。检索中涉及的ENAC内的机制和域尚未完全理解。我们对所有ENAC亚基中发现的两个保守的，上覆的域（S/TPPPXYXS/TL）感兴趣：PY（XPPXY）和基于酪氨酸的内吞（YXXL）基序。先前的基序针对通过NEDD4泛素连接酶进行泛素化的通道。已知后一种基序与AP-2复合物的MU2亚基相互作用，该基元靶向由Dynamin介导的网格蛋白涂层的粘液胶粘膜内吞作用。我们将测试这些域是否是模块化的，因此可以分离或它们是否共同起作用。生理信号传导级联（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域降低通道活性的假设。此外，我们将确定完全保守的S/T是否仅在PY之前和YXXL基序内（适合MAPK的共识序列）是影响MAPK的目标，它影响了MAPK影响通道活动和膜水平。 ENAC是一个杂体通道，该通道由3个不同的亚基组成，其通道包含两种或更少类型的活性亚基类型。因此，亚基检索的一种可能结果是仅包含两种类型亚基的同源通道或通道的产生。由于可能无法固定膜ENAC的亚基化学计量法，因此我们还询问MAPK信号通过PY和YXXL结构域是否会更改ENAC亚基化学计量和/或组成以调节通道活动。在这里，我们解决了四个具体目标：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内吞域控制，并为每个域分配显着性； 3）确定生理细胞信号传导是否通过PY和YXXL基序调节通道检索，以及保守S/T的差异磷酸化是否会调节此检索； 4）确定从质膜中检索ENAC亚基是否是协调的。这项研究将提供有关ENAC分子体系结构和调节该重要渠道的关键见解。