A versatile optical tool for studying synaptic GABAA receptor trafficking

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here we describe an optical tool to study synaptic GABAA receptor (GABAAR) dynamics using adaptable fluorescent tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a 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 by 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 measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAAR trafficking.