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 哺乳动物肺中存在Na+通道，包括心尖细胞。 肺泡II型肺泡细胞膜。活性Na+跨膜转运 成人肺泡上皮受激素控制，这一点很重要。 在正常生理和生理状态下调节肺泡液平衡 病理情况。细菌的生化和分子特征 这些重要的离子通道，荷尔蒙参与的机制 这些通道的调节及其参与的病理生理学 高氧性肺损伤和成人呼吸窘迫等过程 综合征(ARDS)，目前尚不清楚。我们建议检验以下假设 哺乳动物肺泡II型细胞(ATII)含有上皮性Na+通道 而这些通道的活动是由后 翻译修饰，包括磷酸化。具体来说，我们 建议：1)检验哺乳动物ATII细胞含有Low的假设 生化分离纯化阿米洛利亲和Na+通道 这种蛋白质的同质性；2)直接检验假设 从ATII细胞纯化的蛋白通过以下方式形成阿米洛利亲和性Na+通道 生化分离纯化该蛋白，使其均一；2)检测 直接假设从ATII细胞提纯的蛋白质形成 重组纯化蛋白对阿米洛利敏感的阳离子通道 转化为平面脂质双分子层；3)鉴定和表征全长 与组成ATII Na+通道的多肽相对应的基因 以验证分离出的蛋白质确实具有离子功能 通道；以及4)检验磷酸化反应和 盐皮质激素影响脑内Na+通道的表达 转录或翻译水平。这其中的一个重要因素 研究表明，生物化学、生理学和分子生物学 ATII细胞中这些通道的特征将被阐明 第一次，对这种重要蛋白质的新探测器将是 已生成。这一研究无论从基础科学还是从理论上都具有重要意义。 临床方面。因为主动的Na+转运起着非常重要的作用 在保持肺泡无液体方面，特别是在病理情况下 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以 最终对高氧性肺的治疗有重要意义 受伤和急性呼吸窘迫综合征。这项研究的结果将为我们提供新的 对ATII钠离子通道的性质及其修饰方式的见解 通过荷尔蒙，并将有助于建立新的合理的方法，通过 受伤的情况可能会减轻。

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.