Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

摘要: 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 actions gamma-aminobutyric acid (GABA), primary inhibitory neurotransmitter in adult brain, through positive allosteric modulation γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting GABAAR synapse gephyrin scaffold following initial sustained BZD exposure vitro vivo. Using immunofluorescence biochemical experiments, found that cultured cortical neurons treated with classical BZD, diazepam (DZP), presented no substantial change surface or synaptic levels γ2-GABAARs. In contrast, both postsynaptic scaffolding protein showed diminished total a single DZP treatment mouse tissue. We further identified enhanced phosphorylation Ser270 increased generation cleavage products. Selective immunoprecipitation from revealed ubiquitination this exposure. To assess novel trafficking responses induced by DZP, employed an N terminal fluorogen-activating peptide (FAP) pH-sensitive green fluorescent (γ2pHFAP). Live-imaging experiments using γ2pHFAP expressing lysosomal targeting GABAARs overall accumulation vesicular compartments response to DZP. fluorescence resonance energy transfer (FRET) measurements between subunits within neurons, reductions clusters subpopulation sensitive receptor. Moreover, simultaneously exchange γ2-GABAARs recovery after photobleaching (FRAP) techniques. Finally provide first proteomic analysis interactome vs. vehicle mice. Collectively, our results indicate elicits down-regulation availability via multiple dynamic processes.