Dataset Information

Photopotentiation of the GABA_A receptor with caged diazepam.

ABSTRACT: As the inhibitory ?-aminobutyric acid-ergic (GABAergic) transmission has a pivotal role in the central nervous system (CNS) and defective forms of its synapses are associated with serious neurological disorders, numerous versions of caged GABA and, more recently, photoswitchable ligands have been developed to investigate such transmission. While the complementary nature of these probes is evident, the mechanisms by which the GABA receptors can be photocontrolled have not been fully exploited. In fact, the ultimate need for specificity is critical for the proper synaptic exploration. No caged allosteric modulators of the GABA_A receptor have been reported so far; to introduce such an investigational approach, we exploited the structural motifs of the benzodiazepinic scaffold to develop a photocaged version of diazepam (CD) that was tested on basolateral amygdala (BLa) pyramidal cells in mouse brain slices. CD is devoid of any intrinsic activity toward the GABA_A receptor before irradiation. Importantly, CD is a photoreleasable GABA_A receptor-positive allosteric modulator that offers a different probing mechanism compared to caged GABA and photoswitchable ligands. CD potentiates the inhibitory signaling by prolonging the decay time of postsynaptic GABAergic currents upon photoactivation. Additionally, no effect on presynaptic GABA release was recorded. We developed a photochemical technology to individually study the GABA_A receptor, which specifically expands the toolbox available to study GABAergic synapses.

SUBMITTER: Sansalone L

PROVIDER: S-EPMC6800354 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Photopotentiation of the GABA<sub>A</sub> receptor with caged diazepam.

Sansalone Lorenzo L Bratsch-Prince Joshua J Tang Sicheng S Captain Burjor B Mott David D DD Raymo Françisco M FM

Proceedings of the National Academy of Sciences of the United States of America 20191001 42

As the inhibitory γ-aminobutyric acid-ergic (GABAergic) transmission has a pivotal role in the central nervous system (CNS) and defective forms of its synapses are associated with serious neurological disorders, numerous versions of caged GABA and, more recently, photoswitchable ligands have been developed to investigate such transmission. While the complementary nature of these probes is evident, the mechanisms by which the GABA receptors can be photocontrolled have not been fully exploited. In ...[more]

PMID: 31575739

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Project description: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 (GABAARs). Here we define key molecular events impacting γ2 GABAAR 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-GABAARs. 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 (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs 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 GABAAR 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-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes.

Project description:BackgroundWithin the GABA(A)-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABA(A)-receptor subtypes.MethodsWe investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABA(A)-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (?(1) selective) and TPA023 (?(2/3) selective). In-vivo binding studies with [(3)H]flumazenil confirmed compounds occupied CNS GABA(A) receptors.ResultsChronic diazepam treatment resulted in tolerance to diazepam's acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam's anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam's hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam's hypothermic, anxiolytic or sedative effects.ConclusionsOUR DATA INDICATE THAT: (i) GABA(A)-?(2)/?(3) subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam's sedative actions needs concomitant activation of GABA(A)-?(1)/GABA(A)-?(5) receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABA(A) ?(1), ?(2) or ?(5) subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABA(A) ?(2), or ?(3) receptors does not engender tolerance development, subtype-selective GABA(A) drugs might constitute a promising class of novel drugs.

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Photopotentiation of the GABA_A receptor with caged diazepam.

Publications

Photopotentiation of the GABA<sub>A</sub> receptor with caged diazepam.

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Photopotentiation of the GABAA receptor with caged diazepam.

Publications

Photopotentiation of the GABA<sub>A</sub> receptor with caged diazepam.

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OmicsDI is part of the ELIXIR infrastructure

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Photopotentiation of the GABA_A receptor with caged diazepam.