Diazepam Accelerates GABA A R Synaptic Exchange and Alters Intracellular Trafficking

Despite 50+ years of clinical use as anxiolytics, anti‐convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma‐aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABA A R). Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2‐GABA A Rs. In contrast, both γ2 and the post‐synaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in vivo . We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen‐activating peptide (FAP) and pH‐sensitive green fluorescent protein (γ2pHFAP). Live‐imaging experiments using γ2pHFAP GABA A R expressing neurons identified enhanced lysosomal targeting of surface GABA A Rs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between α2 and γ2 subunits within a GABA A R in neurons, we identify reductions in synaptic clusters of this subpopulation of surface benzodiazepine sensitive receptor. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2‐GABA A Rs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the benzodiazepine sensitive GABA A R interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down‐regulation of gephyrin scaffolding and BZD sensitive GABA A R synaptic availability via multiple dynamic trafficking processes. Support or Funding Information This work was supported by funding from a National Institute of Health Training Grant (T32GM008424), National Institutes of Health Predoctoral Individual National Research Service Award (F31 MH117839), University of Pittsburgh Pharmacology and Chemical Biology Fellowship (William C. deGroat Neuropharmacology Departmental Fellowship), NARSAD young investigator grant and Pharmacology and Chemical Biology Startup Funds. Mass spectrometry analyses were conducted at the UTHSCSA Institutional Mass Spectrometry Laboratory, supported in part by UTHSCSA and the University of Texas System for purchase of the Orbitrap Fusion Lumos mass spectrometer. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .