Intracellular Calcium Regulated NHE3 Endocytosis

描述(申请人提供)：在胃肠道(GI)，作为消化中神经体液反应的一部分，在餐后状态下调节电子中性钠吸收。肠道刷状缘(BB)Na+/H+交换器NHE3的调节可以解释大多数已知的中性Na+吸收的消化相关变化，以及腹泻疾病中发生的大部分Na+吸收抑制。NHE3是通过其质膜相对于细胞内位置的变化来调节的，这是内吞和/或胞吐速率变化的结果。在小肠，氨基甲胆碱(模拟胆碱能激活引起的餐后变化)引起的细胞内钙([Ca+]i)升高抑制了NHE3的活性40%，并同样降低了NHE3的表面表达。然而，[Ca~(2+)]i调节NHE3在肠上皮细胞中转运的机制尚不清楚。因此，本研究的长期目标是建立在正常消化和腹泻疾病(例如轮状病毒感染)期间发生的高[Ca+]i条件下调节NHE3活性的内吞途径。目前的提案将试图确定，不依赖于笼蛋白的内吞作用是否有助于电子中性钠吸收的一个重要调节因子在肠上皮细胞中的运输。具体目的：我们建议检验这样的假设：(1)抑制笼状蛋白介导的内吞作用(CME)阻止NHE3的结构性内吞作用，而不是卡巴胆碱介导的内吞作用，以及(2)卡巴胆碱介导的NHE3内吞作用是通过脂筏激活的不涉及笼蛋白的CDC42依赖的途径发生的。我们的NHE3内吞作用可能通过多种内吞途径发生的概念是新颖的，我们目前的提议将首次将NHE3内吞作用分离为网状蛋白依赖和网状蛋白非依赖性途径。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从四个维度表征活的Caco-2BBe细胞(极化的肠上皮细胞模型)中的NHE3内吞作用，以及shRNA敲除技术来定量包括CDC42在内的笼蛋白非依赖性内吞作用的关键成分。意义：使用先进的成像技术不仅可以确定NHE3的内吞作用是如何发生的，还可以为研究其他运输蛋白的运输建立一种新的技术，特别是那些存在于上皮细胞顶端区域的运输蛋白。评估升高的[Ca~(2+)]i调节NHE3活性的机制将有助于理解由NHE3活性降低和钠吸收减少引起的腹泻病的机制。此外，确立细胞骨架蛋白非依赖性内吞作用在肠道转运体[Ca~(2+)]i升高的调节中的作用，可以在转运蛋白调节的早期事件中提供重要信息，从而可能导致替代治疗轮状病毒感染、炎症性肠病或先天性钠腹泻等疾病的急性腹泻症状的治疗策略。 与公共卫生相关：在肠道中，NHE3活性的调节导致了消化过程中发生的钠吸收的大部分已知变化，以及腹泻疾病中发生的钠吸收抑制。细胞内钙离子介导的抑制NHE3活性的升高可能是通过一种不依赖于笼蛋白的内吞途径发生的；然而，这种调节的机制还不是很清楚。目前的研究将为理解肠道钠吸收的一种重要调节因子在消化和腹泻疾病中的运输提供新的见解。

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.