Descrizione del progetto
Definizione della cinetica di attivazione delle giunzioni strette
Negli strati epiteliali, le cellule si uniscono tramite giunzioni strette, complessi multiproteici che regolano il trasporto selettivo tra i compartimenti degli organi. Le proteine claudine svolgono un ruolo fondamentale in queste strutture, formando canali di conduttanza altamente selettivi. Le mutazioni nei geni della claudina causano ipomagnesiemia, insufficienza renale e altre malattie umane, sottolineando l’importanza clinica di queste proteine. Il progetto DynChan, finanziato dall’UE, svilupperà una tecnologia innovativa per studiare il meccanismo alla base della regolamentazione dei trasporti attraverso i canali di claudine. Un chip con array di nanopillari impiegherà nanoelettrodi per sondare eventi a canale singolo su molte giunzioni strette e definire la funzione dei pori di claudine. I risultati accelereranno la nostra comprensione della biologia delle giunzioni strette e contribuiranno all’elaborazione di nuovi interventi terapeutici.
Obiettivo
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)Coordinatore
28006 Madrid
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