GABAA receptors and neuroligin 2 synergize to promote synaptic adhesion and inhibitory synaptogenesis.

GABAA receptors (γ-aminobutyric acid-gated receptors type A; GABAARs), the major structural and functional postsynaptic components of inhibitory synapses in the mammalian brain, belong to a family of GABA-gated Cl-/HCO3 - ion channels. They are assembled as heteropentamers from a family of subunits including: α (1-6), ß(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABAARs together with the postsynaptic adhesion protein Neuroligin 2 (NL2) and many other pre- and post-synaptic proteins guide the initiation and functional maturation of inhibitory GABAergic synapses. This study examined how GABAARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABAAR subtypes, the synaptic type α2ß2γ2-GABAARs versus extrasynaptic type α4ß3δ-GABAARs were expressed in HEK293 cells alone or together with NL2 and co-cultured with striatal GABAergic medium spiny neurons to enable innervation of HEK293 cells by GABAergic axons. When expressed alone, only the synaptic α2ß2γ2-GABAARs induced innervation of HEK293 cells. However, when GABAARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2ß2γ2-GABAAR/NL2 showing a significantly greater synaptogenic activity than α4ß3δ-GABAAR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABAAR subunits and NL2 were co-expressed, and the degree of innervation and synaptic activity assessed, revealing a key role of the γ2 subunit. In biochemical assays, the interaction between NL2 and α2ß2γ2-GABAAR was established and mapped to the large intracellular domain of the γ2 subunit.

Control of cell surface expression of GABAA receptors by a conserved region at the end of the N-terminal extracellular domain of receptor subunits.

Type A γ-aminobutyric acid receptors (GABAARs) represent a family of pentameric GABA-gated Cl-/HCO3- ion channels which mediate inhibitory transmission in the central nervous system. Cell surface expression of GABAARs, a prerequisite for their function, is dependent on the appropriate assembly of the receptor subunits and their transient interactions with molecular chaperones within the endoplasmic reticulum (ER) and Golgi apparatus. Here, we describe a highly conserved amino acid sequence within the extracellular N-terminal domain of the receptor subunits adjoining the first transmembrane domain as a region important for GABAAR processing within the ER. Modifications of this region in the α1, ß3, and γ2 subunits using insertion or site-directed mutagenesis impaired GABAAR trafficking to the cell surface in heterologous cell systems although they had no effect on the subunit assembly. We found that mutated receptors accumulated in the ER where they were shown to associate with chaperones calnexin, BiP, and Grp94. However, their surface expression was increased when ER-associated degradation or proteosome function was inhibited, while modulation of ER calcium stores had little effect. When compared to the wt, mutated receptors showed decreased interaction with calnexin, similar binding to BiP, and increased association with Grp94. Structural modeling of calnexin interaction with the wt or mutated GABAAR revealed that disruption in structure caused by mutations in the conserved region adjoining the first transmembrane domain may impair calnexin binding. Thus, this previously uncharacterized region plays an important role in intracellular processing of GABAARs at least in part by stabilizing their interaction with calnexin.