MOLECULAR MECHANISMS OF EPITHELIAL SODIUM CHANNEL REGULATION

上皮钠通道调节的分子机制

• 批准号: 6110576
• 负责人: CECILIA M CANESSA
• 金额: $ 10.91万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110576
• 关键词: Xenopus oocyte; aldosterone; aminoacid; enzyme activity; epithelium; gene expression; gene mutation; hormone regulation /control mechanism; hypertension; immunofluorescence technique; laboratory rabbit; laboratory rat; molecular cloning; phosphorylation; posttranslational modifications; potassium channel; protein kinase; protein protein interaction; protein structure function; renal tubular transport; salt intake; tissue /cell culture; transcription factor; transfection

Maintenance and variations in blood pressure levels are highly dependent on the total Na+ reabsorbed from the surrounding milieu. The amiloride- sensitive epithelial Na+ channel plays a fundamental role in determining the net amount of Na+ reabsorbed and thus, it is the target for many regulatory mechanisms. This proposal focuses on several aspects of the functional regulation of the amiloride-sensitive epithelial Na+ channel. The aims we are going to pursue are the following: AIM 1. Biosynthesis of Na+ channels and regulation by aldosterone. Effects of aldosterone and of salt intake in the biosynthesis of Na+ channels in the whole animal (in vivo) and in primary cultures of cortical collecting tubules (in vitro). Aldosterone effects will be examined at several time points over a time course in both the in vivo and the in vitro models. The following levels on the biosynthesis of channels will be examined:a) Transcriptional activation of the individual subunits. b)Synthesis of new channel proteins: rate of synthesis, degradation and lifetimes of the individual subunits. c)Assembly of the channel complex, distribution in different cellular compartments and cell surface expression. AIM 2. Modulation of channel activity by phosphorylation. To demonstrate biochemically protein phosphorylation and to identify the cellular pathways and kinases that mediate channel phosphorylation. a)To determine the subunit(s) that are phosphorylated and to identify the amino acids that are phosphorylated by specific kinases. b)To study the changes in channel activity induced by phosphorylation by proteinkinases (PKA, PKC, Ca2+/calmoduline, kinase, thyrosine kinases). c)Functional consequences of replacing the phosphorylated amino acids by other residues. AIM 3. Isolation of other proteins that associate with the Na+ channel. Ion channels are complex multimeric proteins with a general structure consisting of pore-forming subunit(s) and other associated proteins involved in the modulation of channel activity. Here we propose: a)To isolate and identify proteins associated to the subunits of the Na+ channel. b)To examine the functional roles of these proteins in modulating the activity of channels. c)To investigate aldosterone effects on the levels of expression of the associated proteins. d)To study the molecular determinants of the protein- protein interactions and the cellular processes that change these interaction.

血压水平的维持和变化高度依赖 从周围环境中重新吸收的 Na+ 总量。阿米洛利—— 敏感的上皮 Na+ 通道在确定 Na+重吸收的净量，因此，它是许多人的目标 监管机制。该提案主要关注以下几个方面 阿米洛利敏感上皮 Na+ 通道的功能调节。 我们要追求的目标如下： AIM 1. 生物合成 Na+ 通道和醛固酮的调节。醛固酮的作用 整个动物 Na+ 通道生物合成中的盐摄入量（在 体内）和皮质集合管的原代培养物（体外）。 将在一段时间内的几个时间点检查醛固酮的影响 体内和体外模型中的过程。以下级别 将检查通道的生物合成：a) 转录 各个亚基的激活。 b)新通道的合成 蛋白质：合成率、降解率和个体的寿命 亚单位。 c) 通道综合体的组装，分布在不同的地方 细胞区室和细胞表面表达。目标 2. 调制 磷酸化通道活性。证明蛋白质的生化作用 磷酸化并鉴定细胞途径和激酶 介导通道磷酸化。 a) 确定亚基 磷酸化并鉴定被磷酸化的氨基酸 特定激酶。 b) 研究通道活性的变化 蛋白激酶（PKA、PKC、Ca2+/钙调蛋白、激酶、 甲状腺氨酸激酶）。 c) 更换的功能后果 氨基酸被其他残基磷酸化。目标 3. 隔离其他人 与 Na+ 通道相关的蛋白质。离子通道很复杂 具有由成孔组成的一般结构的多聚体蛋白质 亚基和其他参与调节的相关蛋白 渠道活动。在这里我们建议：a）分离和鉴定蛋白质 与 Na+ 通道的亚基相关。 b) 检查功能 这些蛋白质在调节通道活性中的作用。 c) 到 研究醛固酮对表达水平的影响 相关蛋白质。 d) 研究蛋白质的分子决定因素- 蛋白质相互作用和改变这些的细胞过程 相互作用。