FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor

Abstract

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABA_B, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABA_B receptor remains poorly understood. Here, we have developed FRET-based conformational GABA_B sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABA_B receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

Keywords: ACP tag; SNAP tag; allostery; biased ligand; biosensors; lanthanide-based resonance energy transfer LRET; molecular pharmacology; signal transduction; time-resolved FRET.