Recent experimental evidence suggests that amiloride-sensitive epithelial Na+ channels are present in the mammalian lung, including the apical membrane of the alveolar type II pneumocyte. Active Na+ transport across the adult alveolar epithelium is under hormonal control, and is important in regulating alveolar fluid balance under normal physiological and pathological conditions. The biochemical and molecular characteristics of these important ion channels, the mechanisms involved in hormonal modulation of these channels, and their involvement in pathophysiological processes, such as hyperoxic lung injury and Adult Respiratory Distress Syndrome (ARDS), are not known. We propose to test the hypotheses that mammalian alveolar type II cells (ATII) contain epithelial Na+ channels and that the activity of these channels are regulated by post- translational modifications including phosphorylation. Specifically, we propose to: 1) test the hypothesis that mammalian ATII cells contain low amiloride affinity Na+ channels by biochemically isolating and purifying this protein to homogeneity; 2) test directly the hypothesis that the protein purified from ATII cells forms amiloride affinity Na+ channels by biochemically isolating and purifying this protein to homogeneity; 2) test directly the hypothesis that the protein purified from ATII cells forms amiloride-sensitive cation channels by reconstituting the purified protein into planar lipid bilayers; 3) identify and characterize full length cDNA's corresponding to polypeptides comprising the ATII Na+ channel in order to verify that the protein isolated indeed functions as an ion channel; and 4) examine the hypothesis that phosphorylation reactions and mineralocorticoid hormones influence Na+ channel expression at the transcriptional or translational levels. An important element of this study is that the biochemical, physiological, and molecular biological characteristics of these channels in ATII cells will be elucidated for the first time, and that new probes for this important protein will be generated. This research is important both from the basic science and clinical aspects. Because active Na+ transport plays a very important role in maintaining alveoli free of fluid, especially under pathological conditions in which the pulmonary surfactant system has been compromised and surface tension increases, the information obtained from this work may eventually have important implications in the treatment of hyperoxic lung injury and ARDS. The results obtained from this study will offer new insights as to the nature of ATII Na+ channels, the way they are modified by hormones, and will help establish new rational approaches by which lung injury may be alleviated.
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