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.