GABAAR isoform and subunit structural motifs determine synaptic and extrasynaptic receptor localisation
Introduction
γ-aminobutyric acid type-A receptors (GABAARs) form part of the pentameric ligand-gated ion channel family and are the main inhibitory neurotransmitter receptors in the central nervous system (Smart, 2015, Luscher et al., 2011). These receptors are crucial for maintaining control over excitability and neural network computation (Klausberger et al., 2002) and their dysfunction is associated with several neurological conditions including: epilepsy (Oyrer et al., 2018, Macdonald et al., 2010), cognitive impairment (Rudolph and Mohler, 2014), and schizophrenia (Braat and Kooy, 2015, Schmidt and Mirnics, 2015). GABAARs mediate two distinct types of inhibition in the brain: phasic and tonic. Phasic inhibition is brief in duration (typically milliseconds) and arises due to release of GABA from presynaptic interneurons. Activation of postsynaptic GABAARs briefly hyperpolarises and electrically shunts the neuronal membrane to regulate excitability (Mitchell and Silver, 2003). In contrast, tonic inhibition arises due to the persistent background activity of GABAARs in response to ambient and synaptic GABA spillover (Glykys and Mody, 2007). Tonic inhibition exerts a persistent control over neuronal excitability and is important for setting the gain for spike firing (Mitchell and Silver, 2003).
GABAARs are assembled from 19 subunits (α1–6, β1–3, γ1–3, δ, ρ1–3, ε, π, and θ) and the prototypical pentamer consists of 2α, 2β and a γ/δ subunits (Sieghart and Sperk, 2002). Even though such a large number of subunits can generate considerable diversity for receptor subtypes, interestingly, only certain subunit combinations are thought to predominate depending on brain area, cell type and stage of neurodevelopment (Olsen and Sieghart, 2009). Relative spatial segregation of receptor subtypes is observed in ultrastructural and light microscopy studies, and in studies of phasic and tonic inhibition. Using subunit knock-in/-out mice and pharmacological tools, α1β2/3γ2 and α2β2/3γ2 receptors are localised to inhibitory synapses (Kasugai et al., 2010) where they mediate phasic inhibition, e.g. in hippocampal CA1 pyramidal neurons (Prenosil et al., 2006). By contrast, α5β3γ2, α4β3δ, α1βδ and α1β receptor isoforms mediate tonic inhibition in pyramidal and granule cells (Glykys et al., 2007, Glykys et al., 2008, Stell et al., 2003, Sun et al., 2004, Mortensen and Smart, 2006, Zheleznova et al., 2008, Brickley and Mody, 2012, Thomas et al., 2005).
Although they can be spatially discrete, GABAARs are highly dynamic and diffuse laterally within the neuronal plasma membrane between synaptic and extrasynaptic areas (Thomas et al., 2005, Bogdanov et al., 2006, Bannai et al., 2009). GABAARs are inserted into the plasma membrane at extrasynaptic locations and synaptic receptors access inhibitory postsynaptic microdomains by lateral diffusion. At such specialisations, GABAARs are likely to be anchored by scaffold proteins such as gephyrin (Lorenz-Guertin and Jacob, 2018, Tyagarajan and Fritschy, 2014, Mukherjee et al., 2011) and GARLH-family proteins (Yamasaki et al., 2017) resulting in longer residences for synaptic receptors at inhibitory synapses. Nevertheless, the relative extent of synaptic and extrasynaptic receptor access to the inhibitory synapse, and the mechanisms that selectively prevent accumulation of extrasynaptic receptors at the synapse, are unknown. Presently, we know that α5 subunit-containing receptors interact with the extrasynaptic anchoring protein radixin (Hausrat et al., 2015) and this can retain these receptors at perisynaptic/extrasynaptic locations. However, what restricts other GABAAR subtypes to extrasynaptic membrane domains is unknown.
To address this issue, we incorporated an α-bungarotoxin binding site (BBS) mimotope in the N-terminal extracellular domain of prominent hippocampal synaptic (α1 and α2) and extrasynaptic (α4, α5, and δ) subunits. This tag systematically allows the lateral diffusion of GABAARs in proximity to gephyrin-containing inhibitory synapses to be resolved using single particle tracking with a uniform labelling strategy that is not dependent upon N-terminal receptor subunit-specific antibodies. Our results provide evidence that GABAAR subunit composition, as well as specific subunit intracellular motifs play critical roles in determining the localisation of GABAARs on neuronal surface membranes.
Section snippets
cDNA and constructs
Wild-type α1, α2, α4, α5, δ, γ2L, β2 and β3 in pRK5 vector and eGFP in pEGFP-N1 vector, along with myc-tagged α5 and BBS-tagged α1 have been described previously (Hannan et al., 2015, Hannan and Smart, 2018, Loebrich et al., 2006). Gephyrin-GFP cDNA was provided by J. Kittler (UCL, UK). 13 amino acids encoding for the BBS site were cloned into the N-terminus of α2, α4, α5myc and δ GABAAR subunits using PCR (Table 1) followed by ligation and transformation into competent bacteria and screening.
Inserting the α-bungarotoxin binding site into GABAAR isoforms
The membrane diffusion of synaptic and extrasynaptic GABAAR subtypes was studied using a uniform labelling strategy that involved incorporating a mimotope for the α-bungarotoxin (α-BgTx) binding site (BBS) into the N-terminus of α1 (Hannan and Smart, 2018), α2, α4, α5 and δ GABAA-R subunits (Fig. 1A). Since α-BgTx is a low affinity, low efficacy antagonist of GABAARs (Hannan et al., 2015), we applied low (sub-inhibitory) concentrations of 400 nM and tested the specificity of our labelling
Discussion
The activation of neurotransmitter receptors at synapses is an important determinant of synaptic strength. Equally important are the mechanisms that govern the differential localisation and clustering of receptors since these will determine the cell surface numbers that can be activated by neurotransmitters. For GABAARs, it has become increasingly clear that receptor mobility and the accessibility of anchoring molecules can impact on, and help to tune, GABAergic inhibition (Renner et al., 2012,
Acknowledgments
This work was supported by the MRC and Wellcome Trust.
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- 1
Present address: North Middlesex University Hospital, Sterling Way, London N18 1QX.
- 2
Present address: School of Physiology, Pharmacology and Neuroscience, University of Bristol, University Walk, Bristol BS8 1TD, UK.