Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels

Thomas, Philip; Smart, Trevor G.

doi:10.1002/0471141755.ph1104s59

Cited by 2 publications

(1 citation statement)

References 34 publications

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“…where A is GABA concentration, EC 50 is half-maximal GABA concentration and n is the Hill slope. The macroscopic kinetics of GABA-activated currents were studied in HEK-293 cells by applying 1 mM GABA 57 . The activation rate was the time taken to ascend from 10 - 90% of I max and the deactivation rate was the weighted tau of exponential fits from the point of cessation of GABA application until the baseline was attained.…”

Section: Methodsmentioning

confidence: 99%

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABA_AR epilepsy variants

Hannan

Smart

2021

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GABAA receptors (GABAARs) are key orchestrators of neuronal activity and several GABAAR variants have been linked to genetic neurodevelopmental disorders (NDDs) and epilepsies. Here, we report two variants (Met263Lys, Leu267Ile) in the predominant GABAAR α1 subunit gene (GABRA1) that increase apparent receptor affinity for GABA and confer spontaneous receptor activity. These gain-of-function features are unusual because GABAAR variants are traditionally thought to cause seizures by reducing inhibitory neurotransmission. Both Met263Lys and Leu267Ile increased tonic and spontaneous GABAergic conductances in neurons revealed by competitive inhibition and channel block of GABAARs. Significantly, α1-subunit variant expression in hippocampal neurons also reduced dendritic spine density. Our results indicate that elevated GABAergic signalling can precipitate genetic epilepsies and NDDs. Furthermore, the mechanistic basis may involve the de-compartmentalisation of excitatory inputs due to the removal of dendritic spines. This aberrant structural plasticity can be reversed by the naturally-occurring, therapeutically-tractable, inhibitory neurosteroid, pregnenolone sulphate.

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Section: Methodsmentioning

confidence: 99%

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABA_AR epilepsy variants

Hannan

Smart

2021

Preprint

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Differential Coassembly of α1-GABA_ARs Associated with Epileptic Encephalopathy

et al. 2020

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GABA A receptors (GABA A Rs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren Syndrome identified a frameshifting de novo variant in a major GABA A R gene, GABRA1. This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1 Lys374Serfs*25. Cell surface expression of mutant murine GABA A Rs is severely impaired compared to wild-type, due to retention in the endoplasmic reticulum. Mutant receptors were differentially co-expressed with β3, but not with β2 subunits in mammalian cells. Reduced surface expression was reflected by smaller inhibitory postsynaptic currents, which may underlie the induction of seizures. The mutant does not have a dominant negative effect on native neuronal GABA A R expression since GABA current density was unaffected in hippocampal neurons, even though mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential β subunit expression of GABA A R populations, which profoundly affected receptor function and synaptic inhibition. Significance Statement GABA A Rs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain and their dysfunction underlies many neurological disorders, especially epilepsy. Here we report the characterisation of an α1-GABA A R variant that results in severe epilepsy. The underlying mechanism is structurally unusual, with the loss of part of the α1 subunit transmembrane domain and part-replacement with nonsense residues. This led to compromised and differential α1-subunit cell surface expression with β subunits resulting in severely reduced synaptic inhibition. Our study reveals that diseaseinducing variants can affect GABA A R structure, and consequently subunit assembly and cell surface expression, critically impacting on the efficacy of synaptic inhibition, a property that will orchestrate the extent and duration of neuronal excitability.

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Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels

Cited by 2 publications

References 34 publications

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABA_AR epilepsy variants

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABA_AR epilepsy variants

Differential Coassembly of α1-GABA_ARs Associated with Epileptic Encephalopathy

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Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels

Cited by 2 publications

References 34 publications

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABAAR epilepsy variants

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABAAR epilepsy variants

Differential Coassembly of α1-GABAARs Associated with Epileptic Encephalopathy

Contact Info

Product

Resources

About

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABA_AR epilepsy variants

Inhibitory neurosteroid reverses the dendritic spine disorder caused by gain-of-function GABA_AR epilepsy variants

Differential Coassembly of α1-GABA_ARs Associated with Epileptic Encephalopathy