MOLECULAR PROPERTIES OF NA+ CHANNELS IN LUNG ATII CELLS

最近的实验证据表明，阿米洛利敏感的上皮细胞 Na+通道存在于哺乳动物肺中，包括肺顶 肺泡II型肺泡细胞膜。Na+跨膜主动转运 成年肺泡上皮细胞受激素控制， 在正常生理条件下调节肺泡液体平衡 病理条件。的生化和分子特征 这些重要的离子通道，参与激素调节的机制， 这些通道的调节，以及它们在病理生理学上的参与， 过程，如高氧肺损伤和成人呼吸窘迫 综合征（ARDS），尚不清楚。我们建议测试的假设， 哺乳动物肺泡II型细胞（ATII）含有上皮Na+通道 而这些通道的活动是由后- 翻译修饰包括磷酸化。我们特别 建议：1）测试哺乳动物ATII细胞含有低的 阿米洛利亲和Na+通道的生化分离纯化 该蛋白质的同质性; 2）直接测试的假设， 从ATII细胞纯化的蛋白质通过以下方式形成阿米洛利亲和性Na+通道： 生物化学分离和纯化该蛋白质至同质; 2）测试 从ATII细胞纯化的蛋白质形成 阿米洛利敏感性阳离子通道 形成平面脂质双层; 3）鉴定和表征全长 对应于包含ATII Na+通道的多肽的cDNA， 为了验证分离的蛋白质确实作为离子起作用， 通道;和4）检查磷酸化反应和 盐皮质激素影响Na+通道表达 转录或翻译水平。其中一个重要因素是 生物化学、生理学和分子生物学 ATII细胞中这些通道的特性将被阐明， 这是第一次，这种重要蛋白质的新探针将被 生成的.这项研究从基础科学和 临床方面。由于Na+的主动转运在细胞内起着非常重要的作用 在维持肺泡无液体，特别是在病理性 肺表面活性物质系统受损的情况 和表面张力增加，从这项工作中获得的信息可能 最终在高氧肺的治疗中具有重要意义 损伤和ARDS。从这项研究中获得的结果将提供新的 关于ATII Na+通道的性质，它们被修饰的方式， 并将有助于建立新的合理方法， 可以减轻伤害。

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.