MOLECULAR MECHANISMS OF EPITHELIAL SODIUM CHANNEL REGULATION

血压水平的维持和变化高度依赖于 关于从周围环境中重吸收的总Na+。阿米洛利- 敏感的上皮Na+通道在决定 Na+的净量被重新吸收，因此，它是许多人的目标 监管机制。该提案侧重于以下几个方面 阿米洛利敏感的上皮Na+通道的功能调节。 我们将要追求的目标如下：目标1.生物合成 Na+通道与醛固酮的调节。醛固酮和银杏叶提取物的作用 全动物钠离子通道生物合成中的盐摄入量(in 活体)和皮质集合管的原代培养(体外)。 将在一段时间内的几个时间点检查醛固酮的影响 在体内和体外模型中都有进展。以下级别 关于通道的生物合成将被研究：a)转录 激活各个亚基。B)新渠道的合成 蛋白质：个体的合成、降解和寿命 亚单位。C)组装通道复合体，分布在不同的 细胞室和细胞表面的表达。目标2.调制 通过磷酸化来激活通道。用生物化学方法演示蛋白质 磷酸化，并确定细胞通路和激酶 介导通道磷酸化。A)确定以下亚基(S) 磷酸化，并鉴定被磷酸化的氨基酸 特定的激活酶。B)研究以下因素引起的通道活动性变化 蛋白激酶(PKA，PKC，钙/钙调制素，激酶， 甲状腺激活酶)。C)替换 被其他残基磷酸化的氨基酸。目标3.隔离其他 与钠离子通道相关的蛋白质。离子通道是复杂的 具有由造孔组成的一般结构的多聚体蛋白质 亚基(S)和其他参与调节的相关蛋白 通道活动。在这里，我们建议：a)分离和鉴定蛋白质 与钠离子通道的亚基相关联。B)检查功能 这些蛋白质在调节通道活动中的作用。C)至 研究醛固酮对血管内皮生长因子表达水平的影响 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 互动。

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.