ENaC ASSEMBLY, TRAFFICKING AND DEGRADATION IN LUNG

首席调查项目主任(最后、第一、中间)：道格拉斯·C·伊顿描述：说明应用程序的广泛、长期目标和具体目标，并参考项目与健康的相关性。简明扼要地描述实现这些目标的研究设计和方法。避免总结过去的成就和使用第一人称。此摘要的目的是在脱离应用程序时，作为对拟议工作的简洁和准确的描述。如果申请得到资助，这一描述将成为公开信息。因此，不包括专有/机密信息。不要超过所提供的空间。肺上皮细胞的盐分和水运输对于出生时肺部液体的正常清除以及出生后肺内维持呼吸道表面薄薄的液体层以促进肺内气体交换和粘液纤毛清除来自肺的外来颗粒至关重要。肺泡上皮细胞在调节肺盐和液体平衡中起着关键作用。这是通过溶质在肺泡表面和间隙之间的矢量传输实现的。钠的转运是一个两步过程，包括通过钠通道将钠从管腔移动到上皮细胞内部，然后通过基底侧的钠泵将钠主动挤出到浆膜间隙。很大一部分Na+净吸收可被阿米洛利抑制，由于分子生物学研究证实肺上皮细胞中存在对阿米洛利敏感的上皮Na+通道(ENaC)亚单位c_13和7，一般认为这部分Na+转运是由某种形式的ENaC介导的。功能性上皮钠通道(ENaC)是由三个亚基组装成一个四聚体结构形成的。这一过程可能发生在内质网(ER)内，效率低下，因为只有一小部分新合成的ENaC亚单位组装成通道，离开内质网到达质膜。初步实验表明，转导ENaC亚基的细胞和天然上皮细胞都能表达三种不同类型的阳离子通道，这些阳离子通道能够转运Na+。这些高选择性、中等选择性和非选择性的阳离子通道似乎由不同的ENaC亚基组合组成。因此，与可兴奋组织中的多聚体阳离子通道是共翻译组装的不同，阿米洛利敏感的阳离子通道似乎是翻译后组装的，在允许的条件下，一些但不是所有的亚基都可以运输到质膜。这就自然地提出了一系列问题：单个ENaC亚基是如何运输的，亚基在哪里组装成功能离子通道，它们如何插入表面膜，以及它们如何被回收和降解或回收。此外，第二组问题是如何调节这些过程，以产生激素诱导的顶膜功能通道的数量和类型的变化。这些问题构成了这个项目的假设和具体目标的基础。ENaC如何组装、插入和从肺泡细胞膜中移除，将决定肺泡细胞转运Na的能力；因此，对肺生理和病理都有重要意义。表演网站========================================Section End===========================================

¿ Principal InvestigatodProgram Director (Last, first, middle): Douglas C. Eaton DESCRIPTION: State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This abstract is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THF SPACE PROVIDED. Salt and water transport by lung epithelial cells is critical for normal clearance of fluid from the lungs at birth and, in the post-natal lung, for maintaining a thin fluid layer on the surface of the airways to promote pulmonary gas exchange and mucociliary clearance of foreign particulates from the lung. Alveolar epithelial cells play a key role in this regulation of lung salt and fluid balance. This is achieved through vectorial transport of solutes between the alveolar surface and the interstitial spaces. Transport is by a two step process involving movement of sodium from the lumen into the epithelial cell interior through sodium channels and subsequent active extrusion of sodium into the serosal space by the basolateral sodium pump. A significant portion of the net Na + absorption can be inhibited by amiloride, and since molecular biological studies have confirmed the presence of amiloride-sensitive epithelial Na + channels (ENaC) subunits, c_, 13,and 7, in lung epithelia, it is generally assumed that this portion of the Na +transport is mediated by some form of ENaC. Functional epithelial sodium channels (ENaC) are formed by the assembly of some combination of the three subunits into a tetrameric structure. This process presumably occurs within the endoplasmic reticulum (ER) and is inefficient, as only a fraction of newly synthesized ENaC subunits assemble into channels that exit the ER and reach the plasma membrane. Preliminary experiments show that both cells transfected with ENaC subunits and native epithelial cells can express three distinct types of cation channels that are capable of transporting Na +. These highly selective, medium-selective, and non-selective cation channels appear to be composed of different combinations of ENaC subunits. Thus, unlike multimeric cation channels in excitable tissues which are cotranslationally assembled, amiloride-sensitive cation channels appear to be post-translationally assembled and under the permissive conditions some, but not all subunits can traffic to the plasma membrane. This raises the natural set of questions of how individual ENaC subunits are trafficked, where the subunits are assembled into functional ion channels, how they are inserted into the surface membrane, and how they are retrieved and degraded or recycled. In addition, a second set of questions is how these processes are regulated to produce the hormone-induced changes in the number and type of functional channels in the apical membrane. These questions form the basis for the hypotheses and specific aims of this project. How ENaC is assembled, inserted, and removed from alveolar call membranes will determine the capacity of the alveolar cells to transport Na; and, therefore has important implications for both lung physiology and pathology. PERFORMANCE SITE ========================================Section End===========================================