ENaC ASSEMBLY, TRAFFICKING AND DEGRADATION IN LUNG

描述（由申请人提供）：肺上皮细胞的盐和水运输对于出生时肺部液体的正常清除至关重要，并且在出生后的肺中，对于维持气道表面的薄液体层以促进肺部气体交换和肺粘膜纤毛清除肺部外来颗粒至关重要。肺泡上皮细胞在肺盐和液体平衡的调节中起关键作用。这是通过溶质在肺泡表面和间隙之间的矢量运输来实现的。钠转运是一个两步过程，包括钠通过钠通道从管腔进入上皮细胞内部，随后钠通过基底侧钠泵主动挤压进入浆膜空间。阿米洛利可以抑制很大一部分净Na+吸收，由于分子生物学研究已经证实了阿米洛利敏感上皮Na+通道（ENaC）亚基的存在，,吗?然后呢?在肺上皮中，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）是由三种亚基组合成四聚体结构而形成的。这一过程可能发生在内质网（ER）内，并且效率低下，因为只有一小部分新合成的ENaC亚基聚集到通道中，离开内质网并到达质膜。初步实验表明，转染ENaC亚基的细胞和天然上皮细胞都能表达三种不同类型的能够运输Na+的阳离子通道。这些高选择性、中等选择性和非选择性阳离子通道似乎由ENaC亚基的不同组合组成。因此，与可兴奋组织中的多聚体阳离子通道（共翻译组装）不同，酰胺敏感阳离子通道似乎是翻译后组装的，在允许的条件下，一些亚基（但不是所有亚基）可以运输到质膜。这就提出了一系列自然的问题：单个ENaC亚基是如何运输的，亚基在哪里组装成功能性离子通道，它们是如何插入表面膜的，以及它们是如何被回收、降解或再循环的。此外，第二个问题是这些过程是如何被调节以产生激素诱导的顶端膜功能通道的数量和类型的变化的。这些问题构成了这个项目的假设和具体目标的基础。ENaC如何从肺泡膜上组装、插入和移除将决定肺泡细胞运输Na的能力；因此对肺生理学和病理学都有重要意义。

DESCRIPTION (provided by applicant): 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, ?, ? and ? 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.