Intracellular Calcium Regulated NHE3 Endocytosis

描述（由申请方提供）：在胃肠道（GI）中，餐后状态期间电中性Na+吸收作为消化中神经体液反应的一部分受到调节。肠刷状缘（BB）Na+/H+交换器NHE 3的调节解释了大多数公认的中性Na+吸收的消化相关变化，以及大多数在消化性疾病中发生的Na+吸收抑制。NHE 3受其质膜相对于细胞内位置的变化调节，这是内吞和/或胞吐速率改变的结果。在小肠中，卡巴胆碱引起的细胞内钙（[Ca 2 +]i）升高（模拟胆碱能激活引起的餐后变化）抑制NHE 3活性40%，并类似地降低NHE 3表面表达。然而，负责[Ca 2 +]i调节肠上皮细胞中NHE 3运输的机制还不清楚。因此，本研究的长期目标是建立内吞途径，该途径在正常消化过程中以及腹泻疾病（例如轮状病毒感染）期间发生的[Ca 2 +]i升高条件下调节NHE 3活性。目前的建议将试图确定是否网格蛋白独立的内吞作用有助于运输的一个重要的调节剂的电中性钠吸收在肠上皮细胞。具体目标：我们建议检验以下假设：（1）抑制网格蛋白介导的内吞作用（CME）可阻止NHE 3的组成性内吞作用，但不能阻止卡巴胆碱介导的内吞作用;（2）卡巴胆碱介导的NHE 3内吞作用通过脂筏、激活的Cdc 42依赖性途径（不涉及网格蛋白）发生。我们认为NHE 3内吞作用可能通过多种内吞途径发生的概念是新颖的，我们目前的建议将是第一个将NHE 3内吞作用分为网格蛋白依赖性和网格蛋白非依赖性途径的建议。我们建议使用先进的成像技术，包括旋转圆盘共聚焦显微镜来表征活Caco-2BBe细胞（极化肠上皮细胞模型）中四维的NHE 3内吞作用，以及shRNA敲低技术来定量网格蛋白非依赖性内吞作用的关键组分，包括Cdc 42。重要性：使用先进的成像技术不仅可以确定NHE 3内吞作用是如何发生的，而且还可以建立一种新的技术来研究其他转运蛋白的运输，特别是那些存在于上皮细胞顶端结构域的转运蛋白。评估负责通过升高[Ca 2 +]i调节NHE 3活性的机制应有助于理解负责由NHE 3活性降低和钠吸收减少引起的结肠炎疾病的机制。此外，确定网格蛋白非依赖性内吞作用在肠道转运蛋白[Ca 2 +]i调节升高中的作用，可以为转运蛋白调节的早期事件提供重要信息，这可能导致用于治疗包括轮状病毒感染、炎性肠病或先天性钠腹泻在内的疾病的急性腹泻症状的替代治疗策略。 公共卫生相关性：在肠道中，NHE 3活性的调节解释了在消化期间发生的钠吸收的大多数公认变化以及在消化性疾病中发生的钠吸收的抑制。细胞内钙介导的NHE 3活性抑制升高可能通过非网格蛋白依赖性的内吞途径发生;然而，这种调节的机制尚不清楚。目前的研究将提供新的见解，了解运输的一个重要调节肠道钠吸收消化以及在消化性疾病。

DESCRIPTION (provided by applicant): In the gastrointestinal (GI) tract, electroneutral Na+ absorption is regulated during the postprandial state as part of the neurohumoral response in digestion. Regulation of the intestinal brush border (BB) Na+/H+ exchanger, NHE3, accounts for most of the recognized digestion related changes in neutral Na+ absorption, as well as most of the inhibition of Na+ absorption that occurs in diarrheal diseases. NHE3 is regulated by changes in its plasma membrane versus intracellular location as a result of alterations in the rates of endocytosis and/or exocytosis. In the small intestine, elevation of intracellular calcium ([Ca2+]i) by carbachol (mimics postprandial changes due to cholinergic activation) inhibits NHE3 activity by 40% and similarly decreases NHE3 surface expression. However, the mechanisms responsible for [Ca2+]i regulated NHE3 trafficking in intestinal epithelial cells are not well understood. Therefore, the long-term objective of the current study is to establish the endocytic pathway that regulates NHE3 activity under elevated [Ca2+]i conditions which occur during normal digestion as well as during diarrheal diseases (e.g. rotavirus infection). The current proposal will attempt to identify whether clathrin-independent endocytosis contributes to the trafficking of an important regulator of electroneutral sodium absorption in intestinal epithelial cells. SPECIFIC AIMS: We propose to test the hypothesis that (1) inhibition of clathrin-mediated endocytosis (CME) prevents constitutive, but not carbachol-mediated, endocytosis of NHE3, and (2) carbachol-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. Our concept that NHE3 endocytosis may occur through multiple endocytic pathways is novel and our current proposal will be the first to separate NHE3 endocytosis into clathrin-dependent and clathrin- independent pathways. We propose to use advanced imaging techniques including spinning disk confocal microscopy to characterize NHE3 endocytosis in four dimensions in live Caco-2BBe cells (polarized intestinal epithelial cell model) as well as shRNA knockdown technology to quantitate the critical components of clathrin- independent endocytosis, including Cdc42. SIGNIFICANCE: The use of advanced imaging will not only define how NHE3 endocytosis occurs but could also establish a new technique for studying the trafficking of other transport proteins, particularly those that exist in the apical domain of epithelial cells. Evaluating the mechanisms responsible for the regulation of NHE3 activity by elevated [Ca2+]i should contribute to an understanding of a mechanism responsible for diarrheal diseases that result from decreased NHE3 activity and reduced sodium absorption. Furthermore, establishing a role for clathrin-independent endocytosis in elevated [Ca2+]i regulation of intestinal transporters could provide important information in early events of transport protein regulation that could lead to alternate therapeutic strategies for treating acute diarrheal symptoms in diseases including rotavirus infection, inflammatory bowel disease, or congenital sodium diarrhea. PUBLIC HEALTH RELEVANCE: In the intestine, regulation of NHE3 activity accounts for most of the recognized changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs in diarrheal diseases. Elevated intracellular calcium-mediated inhibition of NHE3 activity may occur through an endocytosis pathway that is not clathrin-dependent; however, the mechanism for this regulation is not well understood. The current study will provide novel insights into understanding the trafficking of an important regulator of intestinal sodium absorption in digestion as well as in diarrheal diseases.