Dataset Information

Zolpidem and eszopiclone prime ?1?2?2 GABAA receptors for longer duration of activity.

ABSTRACT:

Background and purpose

GABAA receptors mediate neuronal inhibition in the brain. They are the primary targets for benzodiazepines, which are widely used to treat neurological disorders including anxiety, epilepsy and insomnia. The mechanism by which benzodiazepines enhance GABAA receptor activity has been extensively studied, but there is little mechanistic information on how non-benzodiazepine drugs that bind to the same site exert their effects. Eszopiclone and zolpidem are two non-benzodiazepine drugs for which no mechanism of action has yet been proposed, despite their clinical importance as sleeping aids. Here we investigate how both drugs enhance the activity of ?1?2?2 GABAA receptors.

Experimental approach

We used rapid ligand application onto macropatches and single-channel kinetic analysis to assess rates of current deactivation. We also studied synaptic currents in primary neuronal cultures and in heterosynapses, whereby native GABAergic nerve terminals form synapses with HEK293 cells expressing ?1?2?2 GABAA receptors. Drug binding and modulation was quantified with the aid of an activation mechanism.

Key results

At the single-channel level, the drugs prolonged the duration of receptor activation, with similar KD values of ?80?nM. Channel activation was prolonged primarily by increasing the equilibrium constant between two connected shut states that precede channel opening.

Conclusions and implications

As the derived mechanism successfully simulated the effects of eszopiclone and zolpidem on ensemble currents, we propose it as the definitive mechanism accounting for the effects of both drugs. Importantly, eszopiclone and zolpidem enhanced GABAA receptor currents via a mechanism that differs from that proposed for benzodiazepines.

SUBMITTER: Dixon CL

PROVIDER: S-EPMC4507157 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Zolpidem and eszopiclone prime α1β2γ2 GABAA receptors for longer duration of activity.

Dixon Christine L CL Harrison Neil L NL Lynch Joseph W JW Keramidas Angelo A

British journal of pharmacology 20150511 14

<h4>Background and purpose</h4>GABAA receptors mediate neuronal inhibition in the brain. They are the primary targets for benzodiazepines, which are widely used to treat neurological disorders including anxiety, epilepsy and insomnia. The mechanism by which benzodiazepines enhance GABAA receptor activity has been extensively studied, but there is little mechanistic information on how non-benzodiazepine drugs that bind to the same site exert their effects. Eszopiclone and zolpidem are two non-ben ...[more]

PMID: 25817320

Similar Datasets

Project description:Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)R?2(77I)lox) in which the ?2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (?2(77I)) and used viral vectors to swap ?2(77I) with wild-type, zolpidem-sensitive ?2 subunits (?2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced ?2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged ?2 subunits, with which cortical cultures of GABA(A)R?2(?/?) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged ?2 ((AU1)?2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)?2(77F) subunit (Cre-2A-(AU1)?2(77F)) into the neocortex of GABA(A)R?2(77I)lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1)?2(77F) subunits in perisomatic GABAergic synapses. In line with this,miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of ?2(77I) and (AU1)?2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.