Abstract
GABAA receptors (GABAARs) play a crucial role in mediating inhibition in the adult brain. In spite of progress in describing (mainly) the static structures of this receptor, the molecular mechanisms underlying its activation remain unclear. It is known that in the α1β2γ2L receptors, the mutation of the β2E155 residue, at the orthosteric binding site, strongly impairs the receptor activation, but the molecular and kinetic mechanisms of this effect remain elusive. Herein, we investigated the impact of the β2E155C mutation on binding and gating of the α1β2γ2L receptor. To this end, we combined the macroscopic and single-channel analysis, the use of different agonists [GABA and muscimol (MSC)] and flurazepam (FLU) as a modulator. As expected, the β2E155C mutation caused a vast right shift of the dose–response (for GABA and MSC) and, additionally, dramatic changes in the time course of current responses, indicative of alterations in gating. Mutated receptors showed reduced maximum open probability and enhanced receptor spontaneous activity. Model simulations for macroscopic currents revealed that the primary effect of the mutation was the downregulation of the preactivation (flipping) rate. Experiments with MSC and FLU further confirmed a reduction in the preactivation rate. Our single-channel analysis revealed the mutation impact mainly on the second component in the shut times distributions. Based on model simulations, this finding further confirms that this mutation affects mostly the preactivation transition, supporting thus the macroscopic data. Altogether, we provide new evidence that the β2E155 residue is involved in both binding and gating (primarily preactivation).
Highlights
GABAA receptors (GABAARs) are essential mediators of inhibitory neurotransmission in the adult mammalian brain and play a critical role in maintaining the correct balance of excitatory and inhibitory signaling that govern the proper function of the nervous system
Dysfunction of these channels leads to many neurological and psychiatric disorders such as epilepsy, anxiety, insomnia, schizophrenia, and autistic spectrum disorders
Functional GABAARs are heteropentameric channels co-assembling from a repertoire of 20 subunits (Berezhnoy et al, 2007) while the predominant combination in the vertebrate brain consists of two α1, two β2, and one γ2 subunit (Tretter et al, 1997; Farrar et al, 1999; Farrant and Nusser, 2005)
Summary
GABAA receptors (GABAARs) are essential mediators of inhibitory neurotransmission in the adult mammalian brain and play a critical role in maintaining the correct balance of excitatory and inhibitory signaling that govern the proper function of the nervous system. Besides critical involvement of α1F64 from loop D in receptor gating (Szczot et al, 2014; Kisiel et al, 2018), there are some hints (Newell et al, 2004; Mortensen et al, 2014) suggesting that β2E155 residue in loop B, may be involved in channel gating properties, which is clearly manifested by enhancement of the spontaneous activity when mutating it This residue has been previously shown by the homology modeling of GABAAR (Cromer et al, 2002) and ligand docking (Mortensen et al, 2014) to interact with the positively charged moiety of GABA, most probably by anchoring the GABA amino nitrogen end (Zhu et al, 2018). This fact reveals a particular importance of β2E155 residue in a pathophysiological context
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