REGULATION OF SODIUM IN TIGHT EPITHELIA

该计划的长期目标是检查上皮组织中Ton运输的控制和调节。在……里面 特别是，该项目将使用单通道和生化方法来研究阿米洛利的调节。 肾和肺上皮细胞中可阻断的钠通道。这些频道之所以有趣，是因为它们的相关性 运输组织的不同通道之间的独特性，以及这些通道有趣的荷尔蒙调节。 然而，调节这三种通道的机制还没有完全描述。因此，这个项目 将进一步研究调节三种钠转运上皮细胞钠通道的信号级联 使用膜片钳技术并辅以直接生化测量的细胞系。该计划的具体目标 拟议的拨款期限将调查四个调节钠转运的信号级联。目标是(1)进一步 研究异源三聚体G蛋白信号级联对钠通道的调节；具体地说， G蛋白a亚基是直接激活钠通道还是直接激活钠通道 激活与根尖膜内表面密切相关的其他效应分子； 蛋白Py亚基改变ENaC活性？(2)检测小G蛋白信号对钠通道的调节 瀑布。一个小G蛋白K-Ras2A的激活是维持正常ENaC活动所必需的。元素： K-RAS信号级联似乎与顶膜的胞液表面密切相关，因为 级联可以在切除的、由内向外的补丁中被激活。因此，K-RAS的激活机制和 K-RAS激活的信号分子将被检测。(3)肌醇对钠通道的调节作用 脂类和肌醇类脂酶。在切除的、由内向外的斑块中，钠通道需要存在 磷脂酰肌醇-4，5-二磷酸(4，5-PIP2)和A6细胞具有产生4，5-PIP2所需的酶。(4) 研究醛固酮增加钠通道活性的机制。证明了这些信号 以醛固酮激活K-RAS并导致PI-3K介导的3，4，5-PIP3产生的级联反应 涉及磷脂酰肌醇依赖的激酶(PDK1/2)、血清/糖皮质激素依赖的激酶的激活 (SGK)和泛素连接酶Nedd4。确定这些信号分子是通过PI-3-激活而激活的 该4-PIP-5-激酶被激活以产生4，5-PIP2，随后产生3，4，5-PlP3。最后，我们将使用 商业上可用的基因芯片，用于识别新的醛固酮诱导基因。

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.