Tight junction protein-based water transport

本项目主要研究水通过近端肾单位和小肠等上皮细胞紧密连接的运输及其分子基础。解决了几十年来关于细胞旁水运动的相关性甚至是否存在的争论，我们最近证明了一种独特的紧密连接蛋白，claudin-2，形成细胞旁水通道。到目前为止，我们在上皮细胞模型中测量水渗透性的分子生物学/生理学方法是全球独一无二的，使我们能够研究紧密连接介导的水运输的分子决定因素。在这里，我们想要完成三个副主题。(A)克劳丁-2对阳离子和水都形成一个统一的通道，但另外两个形成离子通道的克拉丁-10b和克拉丁-17不能渗透到水中。在这里，问题出现了，克拉丁-15，一种富含于肠道上皮细胞的阳离子通道前体物，是否也起到了水通道的作用。自己的初步数据确实表明了Claudin-15的透水性。在将claudin-15描述为假定的第二紧密连接水通道之后，我们希望通过定点突变来确定决定水通透性的氨基酸。主要的候选者是第一细胞外环(ECL1)内的那些与克劳丁离子通透性有关的基因。(B)Claudin-1缺陷小鼠出生后第一天就会死于皮肤中致命的水分丢失。这一经典发现表明，Claudin-1对水具有不可或缺的密封作用。然而，在我们的手中，claudin-1基因敲除对人和小鼠角质形成细胞的跨上皮水运输没有影响。为了验证不只是claudin-1，而是其他claudins的表达模式是致病的这一假设，我们将在不同claudin设置的细胞系中研究claudin-1表达变化后的水通性。(C)三细胞紧密连接(TTJ)含有一种特定的蛋白质，三细胞蛋白。根据细胞旁屏障缺陷的新概念，在三细胞蛋白丰度降低的情况下，TTJ变得对大分子和较小的溶质具有渗透性。由于目前对溃疡性结肠炎患者活检组织的实验发现三纤维素减少，确定TTJ是否有助于细胞旁水的通透性具有重要的临床意义。我们希望通过模型上皮细胞中三细胞蛋白的过度表达或下调或三细胞蛋白调节蛋白LSR的下调来确定水的渗透性。该项目的总体目标是(I)解决不同紧密连接蛋白在促进或阻碍细胞旁水运输方面的功能，以及(Ii)阐明claudin水通道形成的分子先决条件。总而言之，该项目将导致对健康和疾病中细胞旁上皮水运输的新的机制和功能的深入了解。

This project focuses on water transport through the tight junction of epithelia like proximal nephron and small intestine, and its molecular basis. Resolving a dispute of several decades on the relevance, or even existence, of paracellular water movement, we have recently demonstrated that one distinct tight junction protein, claudin-2, forms paracellular water channels. Our molecular biologic/physiologic method for measuring water permeability in epithelial cell models is worldwide unique so far and enables us to investigate the molecular determinants of tight junction-mediated water transport. Here, we want to accomplish three subtopics. (A) Claudin-2 forms a uniform channel for both, cations and water, but two other ion channel-forming claudins, claudin-10b and claudin-17, are not permeable to water. Here, the question arises whether or not claudin-15, a cation channel-former abundant in intestinal epithelia, also acts as a water channel. Own preliminary data indeed indicate water permeability of claudin-15. After characterizing claudin-15 as a putative second tight junctional water channel, we want to identify the amino acids which determine water permeability using site-directed mutagenesis. Main candidates are those within the first extracellular loop (ECL1) which are involved in claudin ion permeability.(B) Claudin-1-deficient mice die on day one after birth due to a fatal water loss through the skin. This classical finding indicates an indispensable sealing role of claudin-1 for water. However in our hands, claudin-1-knockdown had no effect on transepithelial water transport in human and mouse keratinocytes. In order to test the hypothesis that not claudin-1 alone, but the expression pattern of other claudins are causative, we will investigate water permeability after alteration of claudin-1 expression in cell lines with different claudin settings.(C) The tricellular tight junction (tTJ) contains a specific protein, tricellulin. Upon reduced abundance of tricellulin the tTJ becomes, according to a new concept of a paracellular barrier defect, permeable to macromolecules and smaller solutes. As in current experiments on biopsies from ulcerative colitis patients tricellulin was found decreased, it is of clinical importance to decide whether or not the tTJ contributes to paracellular water permeability. We want to determine water permeability by overexpression or knock-down of tricellulin or of knock-down of a tricellulin-regulating protein, LSR, in model epithelia. The overall goals of this project are (i) to solve the function of different tight junction proteins in facilitating or hindering paracellular water transport and (ii) to elucidate the molecular prerequisites of claudin water channel formation. In conclusion, this project will lead to new mechanistic and functional insights concerning paracellular epithelial water transport in health and disease.