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A versatile optical tool for studying synaptic GABAA receptor trafficking.

Authors
  • Lorenz-Guertin, Joshua M1
  • Wilcox, Madeleine R2
  • Zhang, Ming3
  • Larsen, Mads B4
  • Pilli, Jyotsna1
  • Schmidt, Brigitte F5
  • Bruchez, Marcel P3, 5
  • Johnson, Jon W2
  • Waggoner, Alan S3
  • Watkins, Simon C4
  • Jacob, Tija C6
  • 1 Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
  • 2 Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.
  • 3 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
  • 4 Department of Cell Biology, Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15213, USA.
  • 5 Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
  • 6 Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA [email protected]
Type
Published Article
Journal
Journal of Cell Science
Publisher
The Company of Biologists
Publication Date
Nov 15, 2017
Volume
130
Issue
22
Pages
3933–3945
Identifiers
DOI: 10.1242/jcs.205286
PMID: 29025969
Source
Medline
Keywords
License
Unknown

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 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.

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