Descripción del proyecto
Determinación de la cinética de los canales paracelulares en las uniones intercelulares estrechas
En las capas epiteliales, las células se unen a través de uniones estrechas, que son complejos multiproteicos que regulan el transporte selectivo entre compartimientos de órganos. Las proteínas de la familia de las claudinas desempeñan un papel fundamental en estas estructuras, ya que forman canales iónicos muy selectivos. Las mutaciones en los genes que codifican las claudinas provocan insuficiencia renal y otras enfermedades humanas, lo que pone de manifiesto la importancia clínica de estas proteínas. El proyecto financiado con fondos europeos DynChan desarrollará una tecnología innovadora para estudiar el mecanismo que subyace a la regulación del transporte a través de los canales paracelulares de claudina. Un chip con una matriz de nanopilares empleará nanoelectrodos para evaluar la actividad de un canal único en múltiples uniones estrechas y determinar la función de los poros de claudina. Los resultados mejorarán nuestra comprensión de la biología de las uniones intercelulares estrechas y contribuirán al diseño de nuevos tratamientos terapéuticos.
Objetivo
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)Coordinador
28006 Madrid
España