A versatile optical tool for studying synaptic GABA A receptor trafficking.

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic gamma-aminobutyric acid (GABA) type A receptor (GABAAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABA(A)R gamma 2 subunit tagged with pH-sensitive green fluorescent protein (gamma 2(pH)FAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. gamma 2(pH)FAP GABA(A)Rs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show gamma 2(pH)FAP GABA(A)Rs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using gamma 2(pH)FAP-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 gamma 2(pH)FAP-MG dye approach reveals enhanced GABA(A)R 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 GABA(A)R trafficking.