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）在建立血压中起着重要作用。ENaC活性部分地由其在质膜中的水平设定。ENaC的膜水平反映了组成性递送和调节性取回。ENaC中参与检索的机制和域尚未完全理解。我们感兴趣的是在所有ENaC亚基中发现的两个保守的重叠结构域（S/TPPPxYxS/TL）：PY（xPPxY）和基于酪氨酸的内吞（YxxL）基序。前一个基序通过Nedd 4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mu 2亚基相互作用，其靶向由发动蛋白介导的网格蛋白包被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，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域降低通道活性的假设。此外，我们将确定是否绝对保守的S/T之前的PY和内的YxxL基序，这适合的共识序列的MAPK，是MAPK影响通道活性和膜水平的目标。ENaC是由3个不同亚基组成的异聚体通道，其中通道含有两种或更少类型的活性降低的亚基。因此，亚基回收的一个可能结果是产生同源通道或仅含有两种类型亚基的通道。由于，膜ENaC的亚基化学计量可能不是固定的，我们还问是否MAPK信号通过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，并指定每个结构域的意义; 3）确定生理细胞信号级联是否通过PY和YxxL基序调节通道恢复，以及保守S/T的差异磷酸化是否调节这种恢复;和4）确定ENaC亚单位从质膜的回收是否协调。这项研究将为ENaC的分子结构和这一重要通道的调控提供重要的见解。