GABA_A receptors and neuroligin 2 synergize to promote synaptic adhesion and inhibitory synaptogenesis

GABA_A receptors (γ-aminobutyric acid-gated receptors type A; GABA_ARs), 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), β(1-3), γ(1-3), δ, ε, π, θ and ρ(1-3). GABA_ARs 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 GABA_ARs and NL2 interact with each other to initiate the formation of synapses. Two functionally distinct GABA_AR subtypes, the synaptic type α2β2γ2-GABA_ARs versus extrasynaptic type α4β3δ-GABA_ARs 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 α2β2γ2-GABA_ARs induced innervation of HEK293 cells. However, when GABA_ARs were co-expressed with NL2, the effect on synapse formation exceeded the individual effects of these proteins indicating a synergistic interaction, with α2β2γ2-GABA_AR/NL2 showing a significantly greater synaptogenic activity than α4β3δ-GABA_AR/NL2 or NL2 alone. To investigate the molecular basis of this interaction, different combinations of GABA_AR 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 α2β2γ2-GABA_AR was established and mapped to the large intracellular domain of the γ2 subunit.