MOLECULAR MECHANISMS OF EPITHELIAL SODIUM CHANNEL REGULATION

血压水平的维持和变化高度依赖于 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+重吸收的净量，因此，它是许多人的目标 监管机制。本提案侧重于以下几个方面： 阿米洛利敏感性上皮Na+通道的功能调节。 我们要追求的目标如下：目标1。生物合成 钠离子通道与醛固酮的调节。醛固酮的作用和 盐摄入量在整个动物Na+通道生物合成中的作用（在 体内）和皮质集合小管的原代培养物（体外）。 醛固酮效应将在一段时间内的几个时间点进行检查 在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 激活单个亚基。B）新通道的合成 蛋白质：合成率、降解率和个体寿命 亚单位。c）渠道综合体的组装，在不同的渠道中的分布 细胞区室和细胞表面表达。AIM 2.调制 磷酸化的通道活性。从生物化学上证明蛋白质 磷酸化，并确定细胞途径和激酶， 介导通道磷酸化。a）确定以下子单元： 磷酸化的氨基酸，并鉴定被磷酸化的氨基酸， 特异性激酶B）为了研究由以下引起的通道活性的变化： 通过蛋白激酶（PKA，PKC，Ca 2 +/钙调蛋白，激酶， 甲状腺素激酶）。（c）更换的功能后果 被其他残基磷酸化的氨基酸。AIM 3.分离其他 与Na+通道相关的蛋白质。离子通道很复杂 多聚体蛋白，其一般结构由成孔蛋白组成， 亚基和其他相关蛋白参与调节 渠道活动。在这里，我们提出：a）分离和鉴定蛋白质 与Na+通道的亚基相关。B）检查功能 这些蛋白在调节通道活性中的作用。（c） 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 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.