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Un sensor fluorescente para el GABA y los ligandos sintéticos del receptor GABA (B)

Anastasiya Masharina¹, Luc Reymond, Damien Maurel

¹Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering, NCCR in Chemical Biology, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.

Journal of the American Chemical Society

|October 26, 2012

Resumen

Los investigadores desarrollaron GABA-Snifit, el primer sensor fluorescente ratiométrico para medir el ácido gamma-aminobutírico (GABA) en células vivas. Esta herramienta ofrece alta especificidad y resolución espacio-temporal para el estudio de la señalización GABAérgica.

Área de la Ciencia:

La neurociencia es la neurociencia.
La bioquímica es la bioquímica.
Biología Molecular Biología Molecular

Sus antecedentes:

El ácido gamma-aminobutírico (GABA) es el principal neurotransmisor inhibidor en el sistema nervioso central.
Los métodos existentes para medir el GABA en las células vivas carecen de suficiente resolución espacio-temporal.
Comprender la señalización GABAérgica es crucial para la investigación neurológica.

Objetivo del estudio:

Desarrollar y caracterizar un nuevo sensor fluorescente ratiométrico para GABA.
Para permitir la medición de alta resolución de la concentración de GABA en la superficie de las células vivas.
Proporcionar una herramienta para investigar la dinámica del sistema GABAérgico y las interacciones farmacológicas.

Principales métodos:

Construcción de una proteína de fusión semisintética (GABA-Snifit) que incorpora el receptor GABA ((B), las etiquetas SNAP / CLIP, un fluoróforo sintético y un antagonista fluorescente.
El ensamblaje de GABA-Snifit en la superficie de las células de mamíferos vivos.
Medición de los espectros de emisión de fluorescencia dependientes del GABA (500-700 nm).
Cuantificación de los cambios ratiométricos en la fluorescencia.

Principales resultados:

GABA-Snifit detecta con éxito GABA con alta especificidad y resolución espacio-temporal en las superficies celulares.
El sensor detecta concentraciones de GABA que van desde el micromolar hasta el millimolar.
Se observó un cambio ratiométrico significativo de 1,8 en las células vivas.
GABA-Snifit demostró su utilidad en la cuantificación de las afinidades de unión de los moduladores de los receptores GABA (B).

Conclusiones:

GABA-Snifit es el primer sensor fluorescente ratiométrico para medir el GABA en células vivas.
Este sensor proporciona una herramienta valiosa para la investigación en tiempo real de la neurotransmisión GABAérgica.
GABA-Snifit facilita la investigación sobre las funciones de los receptores GABA y GABA (B) en los sistemas biológicos y los estados de enfermedad.

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