Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse

Abstract

The accumulation of ␥-aminobutyric acid receptors (GABA A Rs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABA A R subtypes are assembled from ␣1-3, ␤, and ␥2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor ␣2 subunit (pH␣2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pH␣2, including other GABA A R subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABA A R binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABA A Rs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABA A Rs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABA A R proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

Identification of an elaborate complex mediating postsynaptic inhibition

“…Comparing this approach to prior reports of the iPSD using affinity purification(19–21), iBioID offers important advantages. Nearly all proteins previously reported to exist at the iPSD were identified.…”

GARLH Family Proteins Stabilize GABAA Receptors at Synapses

“…Further identification of NL interactors and reconstitution of native NL-containing complexes at synapses may ultimately provide a unifying model relating synapse development, synaptic adhesion and neurotransmitter receptor localization. During revision of this manuscript, 174 proteins including LH4 were reported in immunoprecipitants of GFP-tagged α2-containing GABA A R complexes (Nakamura et al, 2016). Among these proteins, 7 proteins (Cul1, Ephexin, KCTD12, Mfn2, mGluR5, PAK5/7) were confirmed as potential direct interactors using a GST pulldown experiment.…”

Role of GABAAR trafficking in the plasticity of inhibitory synapses

“…Nevertheless, a recent proteomic characterization of inhibitory synapses identified novel GABA A R‐associated proteins and showed that the inhibitory postsynaptic densities are more complex than originally thought (Nakamura et al . ). Although the morphology and molecular composition of GABAergic synapses is very different from that observed in excitatory synapses, the main organization principles are conserved to a large extent.…”

“…) (see also Nakamura et al . ). Additional studies allowed the identification of gephyrin interaction motifs in the homologous regions of α1 and α3 (Maric et al .…”