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）：上皮钠通道（ENaC）在建立血压方面发挥着重要作用。 ENaC 活性部分取决于其在质膜中的水平。 ENaC 的膜水平反映了组成型递送和调节性回收。 ENaC 中涉及检索的机制和领域尚未完全了解。我们对所有 ENaC 亚基中发现的两个保守的重叠结构域 (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；已知后一个基序与 AP-2 复合物的 mu2 亚基相互作用，该亚基针对由动力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，或者它们是否协同作用。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 信号传导通过这些模块化检索域降低通道活动的假设。此外，我们将确定 PY 之前和 YxxL 基序内的绝对保守的 S/T（符合 MAPK 的共有序列）是否是影响通道活性和膜水平的 MAPK 的目标。 ENaC 是由 3 个不同亚基组成的异聚通道，其中通道包含两种或更少类型的活性降低的亚基。因此，亚基修复的一种可能结果是产生同聚通道或仅包含两种类型亚基的通道。由于膜 ENaC 的亚基化学计量可能不固定，我们还询问通过 PY 和 YxxL 结构域的 MAPK 信号传导是否会改变 ENaC 亚基化学计量和/或组成以调节通道活性。在这里，我们解决四个具体目标：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 内吞结构域控制，并为每个结构域分配显着性； 3) 确定生理细胞信号级联是否通过 PY 和 YxxL 基序调节通道修复，以及保守 S/T 的差异磷酸化是否调节这种修复； 4) 确定从质膜中恢复 ENaC 亚基是否协调。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。