A versatile optical tool for studying synaptic GABAA receptor trafficking

Journal of Cell Science
Joshua M Lorenz-GuertinTija C Jacob

Abstract

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2pHFAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2pHFAP GABAARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2pHFAP GABAARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2pHFAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2pHFAP-MG dye approach reveals enhanced GABAAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor m...Continue Reading

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Citations

Feb 22, 2020·Traffic·Lydia A Perkins, Marcel P Bruchez
May 5, 2020·Current Protocols in Neuroscience·Jacob P LombardiTija C Jacob
Dec 11, 2019·International Journal of Molecular Sciences·Tiphaine Péresse, Arnaud Gautier
Aug 29, 2018·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Arnaud Gautier, Alison G Tebo
May 14, 2019·Frontiers in Cellular Neuroscience·Joshua M Lorenz-GuertinTija C Jacob
Sep 8, 2018·Frontiers in Cellular Neuroscience·Joshua M Lorenz-GuertinTija C Jacob

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