Intracellular Calcium Regulated NHE3 Endocytosis

描述（由申请人提供）：在胃肠道（GI）中，作为消化过程中神经体液反应的一部分，电中性Na+吸收在餐后状态下受到调节。肠道刷状边界（BB） Na+/H+交换剂NHE3的调控是目前公认的消化相关中性Na+吸收变化的主要原因，也是腹泻疾病中Na+吸收抑制的主要原因。NHE3受其质膜与细胞内位置的变化所调节，这是由于内吞和/或胞吐速率的改变。在小肠中，碳乙醇升高细胞内钙（[Ca2+]i）（模仿胆碱能激活引起的餐后变化）可抑制NHE3活性40%，并同样降低NHE3表面表达。然而，肠上皮细胞中[Ca2+]i调控NHE3运输的机制尚不清楚。因此，本研究的长期目标是建立在正常消化和腹泻疾病（如轮状病毒感染）期间[Ca2+]i升高的情况下调节NHE3活性的内吞途径。目前的提案将试图确定不依赖网格蛋白的内吞作用是否有助于肠上皮细胞中电子中性钠吸收的重要调节因子的运输。具体目的：我们提出验证以下假设：(1)网格蛋白介导的内吞作用（CME）抑制NHE3的组成性内吞作用，而不是碳甾醇介导的内吞作用；(2)碳甾醇介导的NHE3内吞作用是通过脂质筏激活的cdc42依赖途径发生的，该途径不涉及网格蛋白。我们认为NHE3内吞作用可能通过多种内吞途径发生，这一概念是新颖的，我们目前的提议将是第一个将NHE3内吞作用分为网格蛋白依赖途径和网格蛋白独立途径的研究。我们建议使用先进的成像技术，包括旋转盘共聚焦显微镜在四个维度上表征活Caco-2BBe细胞（极化肠上皮细胞模型）的NHE3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成分，包括Cdc42。意义：利用先进的成像技术不仅可以确定NHE3内吞作用是如何发生的，而且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。通过提高[Ca2+]i来评估NHE3活性调控的机制应该有助于理解NHE3活性降低和钠吸收减少导致腹泻疾病的机制。此外，建立不依赖网格蛋白的内吞作用在肠道转运蛋白[Ca2+]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，这可能导致治疗轮状病毒感染、炎症性肠病或先天性钠性腹泻等疾病的急性腹泻症状的替代治疗策略。

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.