REGULATION OF ENAC EXPRESSION BY NONGENOMIC MECHANISMS

非基因组机制对 ENAC 表达的调节

• 批准号: 6413609
• 负责人: CECILIA M CANESSA
• 金额: $ 24.29万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6413609
• 关键词: MDCK cell; Xenopus oocyte; apical membrane; clathrin; density gradient ultracentrifugation; endocytosis; hormone regulation /control mechanism; immunoprecipitation; laboratory rabbit; membrane biogenesis; renal tubular transport; sodium channel; transfection; yeast two hybrid system

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.

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。这 要评估的第一个假设是细胞中 ENaC 的表达 表面受到网格蛋白介导的内吞作用的主动调节， 在基础条件下，在顶端保持少量通道 膜。激素和环境因素可以增加表达 通过改变渠道的内化率。这是 通过修饰位于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 和内吞机制。评估内吞作用对 ENaC 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测定来评估，例如短 电路电流，以及生物化学电流，例如细胞的生物素化 表面通道。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 将进行专门检查。要检验的第二个假设是 新合成通道传输中的速率限制步骤 是亚基的组装。为了评估这个过程，我们将确定 新合成的通道传递至质膜的速率； 哪些亚基组合导致功能通道传递到 质膜；表达不同亚基的细胞中的半衰期 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 亚基相互作用将包括免疫共沉淀实验， 蔗糖梯度沉降和酵母中的双杂交系统。 这些研究将提供对重要机制的见解 ENaC 表达和皮质钠重吸收的调节 集合管和盐敏感性高血压的发病机制。