Tight junction protein-based water transport

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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.