Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

Abstract

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 Rs). Here we define key molecular events impacting γ2 GABA A R and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo . 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 postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. 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 (γ2 pH FAP). Live-imaging experiments using γ2 pH FAP 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 identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Additional time-series experiments revealed the gephyrin regulating kinase ERK was inactivated by DZP at multiple time points. 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 BZD 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.

Neurobiology and Therapeutic Potential of α5-GABA Type A Receptors

“…Mutation of the α5 subunit H105 residue, a key alpha subunit residue required for forming the benzodiazepine binding site with the γ2 subunit, led to repositioning of α5 H105R subunits into the pharmacologically inactive alpha subunit location (Balic et al, 2009). Interestingly, our recent mass spectrometry analysis identified a specific increase in α5βγ2 containing receptors in the cortex following diazepam injection, consistent with benzodiazepine exposure leading to modification of GABA A R composition and potentially drug effects through α5 plasticity (Lorenz-Guertin et al, 2019).…”

Tapering with Pharmaceutical GHB or Benzodiazepines for Detoxification in GHB-Dependent Patients: A Matched-Subject Observational Study of Treatment-as-Usual in Belgium and The Netherlands

“…This difference might be explained by the different working mechanism of BZDs and GHB. Where BZDs primarily affect GABA-A receptors [11], GHB binds to GABA-B and GHB receptors [2]. Withdrawal symptoms of GHB, probably mediated through GABA-B and GHB receptors, might thus not be sufficiently suppressed through BZDs acting through GABA-A.…”

“…In clinical practice, two pharmacological treatment regimens are commonly used to counteract withdrawal symptoms during GHB detoxification: tapering with benzodiazepines (BZDs) and tapering with pharmaceutical GHB. Benzodiazepines have an allosteric effect on GABA-A receptors, resulting in an increased sensitivity for GABA [11]. The benefits of BZDs compared with pharmaceutical GHB are the wide availability in medical settings, low costs and that tapering with BZDs allows patients to directly quit using GHB.…”

Looking for Novelty in an “Old” Receptor: Recent Advances Toward Our Understanding of GABAARs and Their Implications in Receptor Pharmacology

“…One concern associated with long-term usage of benzodiazepines is the development of tolerance based on observations of reduced GABAergic transmission along with altered subunit composition following chronic benzodiazepine treatment ( Uusi-Oukari and Korpi, 2010 ; Jacob et al, 2012 ; Vinkers and Olivier, 2012 ; Lorenz-Guertin et al, 2019 ). The development of benzodiazepine tolerance likely results from decreased GABA A R surface availability due to enhanced inhibition from prolonged drug exposure ( Gallager et al, 1984 ).…”