The Biochemical Anatomy of Cortical Inhibitory Synapses

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.

Section: Discussionsupporting

confidence: 91%

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

Nakamura

Morrow

Modgil

et al. 2016

“…Het ␣1 KO Increased the Total and Surface Expression of the ␣3 Subunit-In addition to ␣1 subunit-containing GABA A Rs, adult wild type cortices also express ␣3 and ␣2 subunit-containing receptors at GABAergic synapses, albeit at lower abundances (42)(43)(44). Therefore, we determined the effects of Het ␣1 KO on total and surface expression of the ␣3 and ␣2 subunits to determine if either of these other synaptic-type ␣ subunits could substitute for ␣1 on the cell surface.…”

Section: Resultsmentioning

confidence: 99%

Altered Cortical GABAA Receptor Composition, Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

Zhou

Huang²,

Ding

et al. 2013

Background: Heterozygous GABRA1 deletion causes absence epilepsy. Results: The deletion modestly decreased cortical GABA A receptor number but also reduced the rate of receptor endocytosis and thus partially compensated for the deletion by increasing the expression of different receptor isoforms. Conclusion: Heterozygous GABRA1 deletion caused novel alterations in cortical GABA A receptor expression and physiology. Significance: These findings reveal mechanisms of cortical disinhibition in absence epilepsy.

“…Proteomic analysis of excitatory postsynaptic sites using a knock-in mouse expressing TAP-tagged PSD-95 identified 118 associated proteins (23). In contrast, proteomic analysis of inhibitory postsynaptic sites using a transgenic mouse expressing the singly tagged Venus-GABA receptor ␣1 identified 18 associated proteins (71). Thus, unlike excitatory postsynaptic complexes, many of which survive isolation with 2% SDS, inhibitory synaptic complexes appear to be particularly labile making it difficult to generate a complete catalogue of components.…”

Section: Discussionmentioning

confidence: 99%

A Combined Transgenic Proteomic Analysis and Regulated Trafficking of Neuroligin-2

Kang

Cassidy

et al. 2014

Background: Brain inhibitory synaptic connections present challenges for molecular identification and imaging. Results: Transgenic mice expressing tagged neuroligin-2 were used to identify associated complexes and image inhibitory synapses in multiple brain regions. Conclusion: Neuroligin-2-associated complexes are enriched in synaptic components, and neuroligin-2 undergoes regulated dynamin-dependent endocytosis and retromer association. Significance: New data and approaches are presented for brain inhibitory synapse proteomics and imaging.