Identification of a Core Amino Acid Motif within the α Subunit of GABAARs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures

Abstract

The fidelity of inhibitory neurotransmission is dependent on the accumulation of g-aminobutyric acid type A receptors (GABA A Rs) at the appropriate synaptic sites. Synaptic GABA A Rs are constructed from a(1-3), b(1-3), and g2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the a2 subunit that promotes the association between GABA A Rs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the a1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the a2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments.

Inhibitory Synapse Formation at the Axon Initial Segment

“…Experiments performed in another mutant mouse, in which residues 360–375 of the α2 subunit are knocked-in to the α1 subunit (the Gabra 1–2 mouse), increases the affinity of the α1 subunit for CB and leads to an increase in α1-GABA A Rs expression at axo-axonic synapses. These data show that residues 360–375 of the α2 subunit are sufficient for GABA A R stabilization at the AIS (Nathanson et al, 2019). Given that CB has a relatively stronger association with the α2 360–375 motif and is present at the AIS, it stands to reason that CB interactions selectively stabilize α2-GABA A Rs at inhibitory AIS synapses.…”

“…In vitro isothermal titration calorimetry showed that the CB SH3 domain preferentially binds the α2 ICD, over either the α1 or α3 ICD, at residues 360–375, suggesting that this ICD motif is integral to GABA A R subtype-specific protein-protein interactions (Hines et al, 2018). Supporting this hypothesis, knocking the α2 360–375 motif into the α1 subunit in mice leads to increased immunoprecipitation of endogenous CB with the chimeric α1 subunit (Nathanson et al, 2019). This same study also showed an increase in the pull-down of GPN with mutant α1, demonstrating a possible synergistic interaction between CB/GPN/α2 ICD that is overall strengthened when the interaction between two partner proteins is enhanced (Nathanson et al, 2019).…”

“…Supporting this hypothesis, knocking the α2 360–375 motif into the α1 subunit in mice leads to increased immunoprecipitation of endogenous CB with the chimeric α1 subunit (Nathanson et al, 2019). This same study also showed an increase in the pull-down of GPN with mutant α1, demonstrating a possible synergistic interaction between CB/GPN/α2 ICD that is overall strengthened when the interaction between two partner proteins is enhanced (Nathanson et al, 2019). The overarching question becomes: does this α2 ICD motif and its preferential protein interactions play a role in subtype-specific synapse formation in the brain, particularly in the construction of α2-enriched synapses at the AIS?…”

Collaborative Cross mice reveal extreme epilepsy phenotypes and genetic loci for seizure susceptibility

“…This result is consistent with recent human and mouse studies that identified GABRA2 contributing to epilepsies. 5,[31][32][33][34] The B6J allele of Gabra2 was also identified as likely responsible for modifying seizure phenotype in a cross between strain B6J and 129S6 of the mouse model of Dravet syndrome. 35,36 Using whole genome sequence from CC027 as well as the reference B6J sequence, we were able to identify founder haplotype and genetic variation in the exons for Gabra2.…”

Gabra2 is a genetic modifier of Scn8a encephalopathy in the mouse*

“…20 Recent studies suggest that the α2 subunit plays a critical role in inhibitory synaptogenesis. 21 Disruption of subcellular localization of the α2 subunit resulted in seizures and early mortality. 22 Strain C57BL/6J carries a hypomorphic splice site variant that reduces Gabra2 transcript level by 75%.…”