REGULATION OF ENAC EXPRESSION BY NONGENOMIC MECHANISMS

上皮性钠通道(ENaC)的表达水平是主要的 远端肾单位净钠重吸收的决定因素。这个 要评估的第一个假设是ENaC在细胞中的表达 表面受细胞骨架蛋白介导的内吞作用的积极调节， 在基本条件下，在顶端维持较少数量的通道 薄膜。激素和环境因素可以增加该基因的表达 通过修改渠道的内部化速度来调整渠道。这是 通过修饰位于其附近的氨基酸来实现 改变通道蛋白之间相互作用的内吞信号 以及内吞机制。评价内吞作用在ENaC中的作用 我们将野生型或突变型通道导入MDCK细胞 缺乏内吞信号。渠道的表达水平和 内吞速率将通过功能分析来评估，如Short- 电路电流和生化电流，如细胞的生物素化 表面渠道。对经络内吞速率的影响 由醛固酮、胰岛素、ADH和鲁米那浓度诱导 将会被特别检查。需要检验的第二个假设是 在传送新合成的频道时的速率限制步骤 是亚单位的组装。为了评估这一过程，我们将确定 新合成的通道向质膜输送的速率； 什么亚基组合导致将功能通道传递到 质膜；亚基在细胞中的半衰期变化 亚基组合，以及参与亚基的结构域 识别和组装。研究的实验方法 亚基的相互作用将包括免疫共沉淀实验， 酵母中蔗糖梯度的沉淀和双杂交系统。 这些研究将提供对以下机制的重要洞察 皮层对ENaC表达和钠重吸收的调节 集合管与盐敏感型高血压的发病机制。

The level of expression of epithelial sodium channels (ENaC) is the main determinant of the net sodium reabsorption by the distal nephron. The first hypothesis to be evaluated is that expression of ENaC at the cell surface is actively regulated by clathrin-mediated endocytosis which, under basal conditions, maintains a low number of channels at the apical membrane. Hormones and environmental factors can increase the expression of channels by modifying the rate of their internalization. This is achieved by modifications of amino acids located in the vicinity of the endocytic signals that change the interaction between the channel proteins and the endocytic machinery. To evaluate the role of endocytosis on ENaC expression we will transfect MDCK cells with wild-type or mutant channels lacking the endocytic signals. The level of expression of channels and the rate of endocytosis will be assessed by functional assays such as short- circuit current, and biochemical ones such as biotinylation of cell surface channels. The effects on the rate of endocytosis of channels induced by aldosterone, insulin, ADH, and luminal sodium concentration will be specifically examined. The second hypothesis to be examined is that the rate limiting step in the delivery of newly synthesized channels is the assembly of subunits. To evaluate this process we will determine the rate of delivery of newly synthesized channels to the plasma membrane; what subunit combinations result in delivery of functional channels to the plasma membrane; the half-life of the subunits in cells expressing varied subunit combinations, and the domains that participate in subunit recognition and assembly. The experimental approaches to investigate subunit interactions will consist on co-immunoprecipitation experiments, sedimentation in sucrose gradients and the two-hybrid system in yeast. These studies will provide insight into mechanisms important to the regulation of ENaC expression and sodium reabsorption by the cortical collecting tubule, and the pathogenesis of salt-sensitive hypertension.