Projektbeschreibung
Definition der Gating-Kinetik von Tight Junctions
Die Zellen in Epithelschichten sind durch Tight Junctions verbunden, Multiproteinkomplexe, die den selektiven Transport zwischen Organkompartimenten regulieren. In diesen Strukturen spielen Claudin-Proteine eine entscheidende Rolle, da sie hoch selektive Ionenkanäle bilden. Mutationen des Claudin-Gens verursachen Hypomagnesiämie, Nierenversagen und andere Krankheiten des Menschen, was die klinische Bedeutung dieser Proteine unterstreicht. Ziel des EU-finanzierten Projekts DynChan ist die Entwicklung einer innovativen Technologie zur Untersuchung des der Transportregulation durch die Claudin-Kanäle zugrundeliegenden Mechanismus. Nanoelektroden werden an einen Nanopillar-Array-Chip angebracht, um Einzelkanalereignisse in vielen Tight Junctions zu analysieren und die Porenfunktion der Claudine zu definieren. Die Ergebnisse werden das Verständnis der Biologie der Tight Junctions verbessern und zur Entwicklung neuartiger therapeutischer Interventionen beitragen.
Ziel
Epithelial paracellular, i.e. tight junction, permeability is largely defined by the integrated functions of claudin proteins that can either seal the paracellular space or form highly-selective conductance channels. The importance of claudins is exemplified by the many human diseases caused by barrier dysregulation and claudin mutations.
The host laboratory recently reported the first measurements of single channel tight junction currents, thereby demonstrating that claudin channels transition between open and closed states. The central hypothesis of this application is that claudin channel activity is regulated by specific molecular interactions.
Unfortunately, the trans-tight junction patch-clamp method developed by the host laboratory is extremely labor intensive and unable to capture more than a small section of a single tight junction, making it unsuitable for comprehensive analyses. To overcome this obstacle, we first aim to develop a nanopillar array chip that will supersede the patch-clamp method. Cells grown over and around the nanopillars will form tight junctions above the nanoelectrode at the tip of each nanopillar. This will make it possible to measure large numbers of single-channel events over many junctions.
The second aim will exploit the nanopillar chip to define the conductances and gating activities of claudin proteins and the mechanisms by which they are regulated. This novel technology will also allow others to analyze claudin function in health and disease. The nanopillar chip and data generated using this tool will accelerate our understanding of tight junction biology and enable development of channel modulators that, in a manner analogous to the advances enabled by transmembrane ion channel modulators, will lead to novel therapeutic approaches.
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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Koordinator
28006 Madrid
Spanien