GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Dixon, Curt B.; Sah, Pankaj; Lynch, Joseph W.; Keramidas, Angelo

doi:10.1074/jbc.m113.514695

Cited by 83 publications

(118 citation statements)

References 58 publications

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“…5 a shows examples of single-channel recordings from BLMs after centrifugation with membrane vesicles containing a1b2g2-type GABA A R at 800 rpm. Stepwise currents were clearly observed with a single-channel conductance of 25 pS, which was close to a previously reported value (25-27 pS) for a1b2g2 GABA A R expressed in HEK 293 cells (45,46). The channel activities were only observed in the presence of GABA (trans side) and were completely blocked by the addition of picrotoxin (trans side), a specific blocker for GABA A R (Fig.…”

Section: Use Of Centrifugal Force To Accelerate Vesicle Fusion To Incsupporting

confidence: 90%

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

Hirano‐Iwata

Ishinari

Yoshida

et al. 2016

Biophysical Journal

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Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Na v 1.5), and the human GABA A receptor (GABA A R) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 Â g). The characteristic channel properties were retained for hERG, Na v 1.5, and GABA A R channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.

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Section: Use Of Centrifugal Force To Accelerate Vesicle Fusion To Incsupporting

confidence: 90%

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

Hirano‐Iwata

Ishinari

Yoshida

et al. 2016

Biophysical Journal

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“…Because it is not possible to control GABA A R subunit composition in neurons, we employed a heterosynapse preparation in which the postsynaptic GABA A Rs comprised only the subunits of interest (32). These recordings were also used to determine the effects of temperature (22, 37 and 40 o C) and anti-epileptic drugs (midazolam and carbamazepine) and on IPSC rise and decay kinetics.…”

Section: A295dmentioning

confidence: 99%

Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy

Chen

Durisic

Lynch

et al. 2017

Neurobiology of Disease

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Epilepsy is a spectrum of neurological disorders with many causal factors. The GABA type-A receptor (GABA A R) is a major genetic target for heritable human epilepsies. Here we examine the functional effects of three epilepsy-causing mutations to the 1 subunit (1 A295D22L receptors also exhibited a heightened temperature sensitivity. In addition, the 1 T10'I22L GABA A Rs were refractory to modulation by carbamazepine or midazolam. In agreement with previous studies, we found that22L GABA A Rs were retained intracellularly in HEK293 cells and neurons. However, preincubation with 100 nM suberanilohydroxamic acid (SAHA) induced 1 A295D22L GABA A Rs to mediate IPSCs that were indistinguishable in magnitude and waveform from those mediated by 122L receptors. Finally, mutation-specific changes to synaptic bouton size, synapse number and neurite branching were also observed. These results provide new insights into the mechanisms of epileptogenesis of 1 epilepsy mutations and suggest possible leads for improving treatments for patients harbouring these mutations.

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“…Finally, site-directed mutagenesis studies of residues in and around the benzodiazepine-binding pocket of α1β2γ2 GABA A receptors suggest the two modulators may bind in different orientations (Hanson et al, 2008) and enhance GABA-mediated currents via structurally different mechanisms (Morlock and Czajkowski, 2011). The aim of the present study is to investigate GABAA receptor-mediated current modulation by eszopiclone and zolpidem within the framework of a data-derived activation mechanism that has previously been validated by us and others (Lema and Auerbach, 2006;Dixon et al, 2014), with the addition of an explicit binding step for the modulator.…”

Section: Introductionmentioning

confidence: 79%

Zolpidem and eszopiclone prime α1β2γ2 GABA_A receptors for longer duration of activity

Dixon

Harrison

Lynch

et al. 2015

British J Pharmacology

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BACKGROUND AND PURPOSEGABAA 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 α1β2γ2 GABAA receptors. EXPERIMENTAL APPROACHWe 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 α1β2γ2 GABAA receptors. Drug binding and modulation was quantified with the aid of an activation mechanism. KEY RESULTSAt 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 IMPLICATIONSAs 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. AbbreviationsAP-5, D-aminophosphonovaleric acid; CNQX, 7-nitro-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-carbonitrile; TTX, tetrodotoxin BJP British Journal of Pharmacology

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GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Cited by 83 publications

References 58 publications

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy

Zolpidem and eszopiclone prime α1β2γ2 GABA_A receptors for longer duration of activity

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GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Cited by 83 publications

References 58 publications

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces

Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy

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

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Zolpidem and eszopiclone prime α1β2γ2 GABA_A receptors for longer duration of activity