REGULATION OF SODIUM IN TIGHT EPITHELIA

该项目的长期目标是研究上皮组织中ton转运的控制和调节。在 特别是，本项目将使用单通道和生化方法来检查阿米洛利的调节， 肾和肺上皮细胞中可阻断的钠离子通道。这些渠道很有趣，因为它们的相对 这是因为在运输组织中的通道之间的独特性，并且因为这些通道的有趣的激素调节。 然而，这些通道的调节机制尚未完全描述。因此本项目 将进一步研究调节三种钠转运上皮细胞中钠通道的信号级联， 使用膜片钳技术辅以直接生物化学测量的细胞系。的具体目标， 建议的资助期间将调查四个信号级联调节钠转运。目标是：（1）进一步 检查钠通道的调节由异三聚体G蛋白信号级联;具体来说，什么是 G蛋白a亚基直接激活Na通道还是直接激活Na通道？ 激活与顶膜内表面密切相关的其他效应分子; 蛋白质Py亚基改变ENaC活性？(2)通过小G蛋白信号检查钠通道的调节 瀑布一种小G蛋白K-Ras 2A的激活是维持正常ENaC活性所必需的。要素 K-Ras信号级联似乎与顶膜的胞质表面密切相关，因为 级联可以在切除的、由内而外的斑块中被激活。因此，K-Ras的激活机制和 将检测由K-Ras激活的信号分子。(3)检查肌醇对钠通道的调节 脂质和肌醇脂质激酶。钠离子通道在切除的，由内而外的补丁需要的存在， 磷脂酰肌醇-4，5-二磷酸（4，5-PIP 2）和A6细胞具有产生4，5-PIP 2所必需的酶。（四） 研究醛固酮增加钠通道活性的机制。证明信号 以K-Ras的醛固酮活化开始并导致PI-3 K介导的3，4，5-PIP 3产生的级联反应 涉及磷脂酰肌醇依赖性激酶（PDK 1/2）、血清/糖皮质激素依赖性激酶 (SGK)和泛素连接酶Nedd 4。确定这些信号分子是通过PI-3的激活而激活的， 激酶和4-PIP-5-激酶被激活以产生4，5-PIP 2和随后3，4，5-PIP 3。最后，我们将使用 市售的基因芯片，以确定新的醛固酮诱导的基因。

The long term goal of this program is to examine the control and regulation of ton transport in epithelial tissue. In particular, this project will use single channel and biochemical methods to examine the regulation of amiloride- blockable sodium channels in renal and lung epithelialcells. These channels are interesting because of their relative uniqueness among channels in transporting tissue and because of the interesting hormonal regulation of these channels. However, the mechanisms for regulation of thee channels have not been completely described. Therefore, this project will further investigate the signaling cascades which regulate sodium channels in three sodium-transporting epithelial cell lines using patch clamp techniques supplemented by direct biochemical measurements. The specific aims for the proposed grant period will investigate four signaling cascades that regulate sodium transport. The aims are (1) further examine the regulation of sodium channels by heterotrimeric G protein signaling cascades;specifically, what is the nature of the interaction between Ga;.3 and EnaC; do the G protein a subunits activate Na channels directly or do they activate some other effector molecule closely associated with the inner surface of the apical membrane; and do G protein Py subunits alter ENaC activity? (2) Examine the regulation of sodium channels by small G protein signaling cascades. The activation of one small G protein, K-Ras2A , is required to sustain normal ENaC activity. Elements of the K-Ras signalingcascade appear to be closely associated with the cytosolic surface of the apical membrane since the cascade can be activated in excised, inside-out patches. Therefore, the mechanism of activation of K-Ras and the signaling molecules activated by K-Ras will be examined. (3) Examine the regulation of sodium channels by inositol lipids and inositol lipid kinases. Sodium channels in excised, inside-out patches require the presence of phosphatidylinositol-4,5-bis-phosphate (4,5-PIP2) and A6 cells have the necessary enzymes to produce 4,5-PIP2. (4) Investigate the mechanisms by which aldosterone increases sodium channel activity. Demonstrate that the signaling cascade that begins with aldosterone activation of K-Ras and leads to the PI-3K-mediated production of 3,4,5-PIP3 involves activation of phosphatidylinositol-dependent kinase (PDK1/2), serum /glucocorticoid-dependent kinsase (SGK), and the ubiquitin ligase, Nedd4. Determine that these signaling molecules are activated by activation of PI-3- kinase and that 4-PIP-5-kinase is activated to produce 4,5-PIP2 and subsequently 3,4,5-PlP3. Finally, we will use commercially available gene chips to identify new aldosterone-induced genes.