REGULATION OF ENAC EXPRESSION BY NONGENOMIC MECHANISMS

上皮钠通道（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。的 待评估的第一个假设是ENaC在细胞中的表达 表面由网格蛋白介导的内吞作用主动调节， 在基础条件下，在顶端保持少量通道 膜的激素和环境因素可增加表达 通过改变通道的内化速率来改变通道的功能。这是 通过修饰位于邻近的氨基酸来实现， 改变通道蛋白之间相互作用的内吞信号 和内吞机制。评价内吞作用在ENaC中的作用 表达，我们将用野生型或突变型通道转染MDCK细胞 缺乏内吞信号渠道的表达水平和 内吞作用的速率将通过功能性测定来评估， 电路电流和生物化学因素，如细胞的生物素化 表面渠道对通道内吞速率的影响 由醛固酮、胰岛素、ADH和鲁米那钠浓度诱导 将进行具体审查。第二个要检验的假设是 在新合成通道的传送中的速率限制步骤 是亚单位的集合。为了评估这一过程，我们将确定 新合成通道向质膜的输送速率; 什么样的亚基组合导致功能性通道的传递 质膜;表达不同的细胞中亚基的半衰期 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 亚基相互作用将由免疫共沉淀实验组成， 蔗糖梯度沉降和酵母双杂交系统。 这些研究将提供重要的机制， 皮层对ENaC表达和钠重吸收的调节 集合小管的变化，以及盐敏感性高血压的发病机制。

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.