A versatile optical tool for studying synaptic GABAA receptor trafficking

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 (GABAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2FAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2FAP GABARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2FAP GABARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2FAP-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 γ2FAP-MG dye approach reveals enhanced GABAR 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 GABAR trafficking.

Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

“…GABA A Rs are ligand-gated ionotropic chloride (Cl -) channels responsible for the majority of fast inhibitory neurotransmission in the adult CNS. The most prevalent synaptic GABA A R subtype is composed of two α, two β, and a γ2 subunit forming a heteropentamer (1). Benzodiazepines (BZD) are a widely used clinical sedative-hypnotic drug class that selectively binds between the interface of a γ2 subunit and either an α1/2/3/5 subunit (2).…”

Fluorogen activating protein toolset for protein trafficking measurements

“…A FAP tool was designed to track multistage synaptic GABA a Receptor internalization and trafficking, extendable for future use in drug related HT screens (Figures B and ) . These receptors function as ligand gated chloride channels involved in inhibitory neurotransmission in the brain .…”

“…They found with neuron exposure to a bicuculline‐induced seizure paradigm that GABA A R had enhanced turnover rates and lysosomal targeting that were not detectable by employing pHluorin (pHGFP) fluorescence alone. pHGFP is quenched in the endosomal‐lysosomal acidic compartments, while γ2pHFAP reports pH of the extracellular environment and throughout the endo‐lysosomal trafficking network . The platform has established a trafficking paradigm of GABA A Rs that can be used to assess trafficking changes caused by different agents.…”

“…A FAP tool was designed to track multistage synaptic GABA a Receptor internalization and trafficking, extendable for future use in drug related HT screens ( Figures 6B and 7). 43 These receptors function as ligand gated chloride channels involved in inhibitory neurotransmission in the brain. 62 GABA a R primarily consist of two subunits, with the γ2 subunit being heavily involved in GABA a R function-and critical for receptor synaptic targeting and cluster maintenance.…”

“…• A versatile optical tool for studying synaptic GABAA receptor trafficking. (Figure 7, dL5**: Cy3 (S/SA)pH-MG) 43 • PI3K class II α regulates δ-opioid receptor export from the trans-golgi network. ( Figure 6D, dL5**: MG-Ester) 44…”