REGULATION OF SODIUM IN TIGHT EPITHELIA

紧密上皮细胞中钠的调节

• 批准号: 7990026
• 负责人: Douglas C. Eaton
• 金额: $ 10万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-15 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990026
• 关键词: 1-Phosphatidylinositol 3-Kinase; Accounting; Adaptor Signaling Protein; Aldosterone; Amiloride; Antisense Oligonucleotides; Apical; Binding; Binding Sites; Biochemical; C-terminal; Cell Line; Cells; Charge; Chimeric Proteins; Chinese Hamster Ovary Cell; Elements; Enzymes; Epithelial; Epithelial Cells; Epithelium; G-substrate; GTP-Binding Proteins; Gene Chips; Genes; Genetic Transcription; Glucocorticoids; Goals; Grant; Heterotrimeric GTP-Binding Proteins; Homologous Gene; Individual; Inositol; Ion Transport; Kidney; Laboratories; Lipids; Lung; MAP Kinase Gene; MAP Kinase Modules; MAPK14 gene; Measurement; Mediating; Membrane; Methods; Methylation; Modeling; Monomeric GTP-Binding Proteins; Mus; Mutate; N-terminal; Nature; P2Y2 receptor; Patch-Clamp Techniques; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase A2; Phospholipase C; Phosphoric Monoester Hydrolases; Phosphotransferases; Potassium; Production; Protein Dephosphorylation; Protein Kinase C; Proteins; Regulation; Relative (related person); Retrieval; Role; Serum; Signal Transduction; Signaling Molecule; Sodium; Sodium Channel; Source; Surface; Technology; Time; Tissues; Translations; apical membrane; base; citrate carrier; epithelial Na+ channel; hormone regulation; inorganic phosphate; interest; kidney cell; member; phosphatidylinositol 3,4,5-triphosphate; potassium ion; programs; protein K; ras Proteins; ubiquitin ligase

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.

该程序的长期目标是检查上皮组织中吨转运的控制和调节。在 特别是，该项目将使用单个通道和生化方法来检查Amiloride-的调节 肾上皮和肺上皮细胞中的可阻滞钠通道。这些频道很有趣，因为它们的亲戚 在运输组织中的通道之间的独特性以及这些通道的荷尔蒙调节有趣。 但是，尚未完全描述调节您通道的调节机制。因此，这个项目 将进一步研究信号级联，该信号级联反应在三个钠传输上的上皮中调节钠通道 使用斑块夹技术补充了直接生化测量的细胞系。具体目标 拟议的赠款期将调查调节钠运输的四个信号级联。目的是（1） 检查异源三聚体G蛋白信号级联对钠通道的调节；具体来说，什么是 GA; .3和ENAC之间相互作用的性质； G蛋白A亚基直接激活Na通道或 激活与顶膜内表面密切相关的其他效应分子；并做g 蛋白质PY亚基改变了ENAC活性？ （2）通过小G蛋白信号传导检查钠通道的调节 级联。需要一个小的G蛋白K-RAS2A的激活才能维持正常的ENAC活性。元素 K-Ras SignalingCascade似乎与根尖膜的胞质表面密切相关，因为 可以用切除的内而外的补丁激活级联反应。因此，K-RAS激活的机理和 将检查由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。切除的内而外贴片中的钠通道需要 磷脂酰肌醇-4,5-双磷酸（4,5-PIP2）和A6细胞具有产生4,5-PIP2的必要酶。 （4） 研究醛固酮增加钠通道活性的机制。证明信号传导 从醛固酮激活K-RAS开始，并导致PI-3K介导的3,4,5-PIP3的产生 涉及磷脂酰肌醇依赖性激酶（PDK1 /2），血清 /糖皮质激素依赖性激酶的激活 （SGK）和泛素连接酶Nedd4。确定这些信号分子是通过激活PI-3-激活的 激酶和4-PIP-5-激酶被激活以产生4,5-PIP2，然后以3,4,5-Plp3产生。最后，我们将使用 可识别新醛固酮诱导的基因的市售基因芯片。