Abstract
GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system. Dysfunction of trafficking and localization of GABAARs to cell membranes is clinically associated with severe psychiatric disorders in humans. The GABARAP protein is known to support the stability of GABAARs in synapses, but the underlying molecular mechanisms remain to be elucidated. Here, we show that GABARAP/GABARAPL1 directly binds to a previously unappreciated region in the γ2 subunit of GABAAR. We demonstrate that GABARAP functions to stabilize GABAARs via promoting its trafficking pathway instead of blocking receptor endocytosis. The GABARAPL1–γ2-GABAAR crystal structure reveals the mechanisms underlying the complex formation. We provide evidence showing that phosphorylation of γ2-GABAAR differentially modulate the receptor’s binding to GABARAP and the clathrin adaptor protein AP2. Finally, we demonstrate that GABAergic synaptic currents are reduced upon specific blockage of the GABARAP–GABAAR complex formation. Collectively, our results reveal that GABARAP/GABARAPL1, but not other members of the Atg8 family proteins, specifically regulates synaptic localization of GABAARs via modulating the trafficking of the receptor.
Highlights
GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system
A recombinant of the TM3–TM4 intracellular domain (ICD) lacking the aforementioned 424–441 octadecapeptide could still bind to GABAAR-associated protein (GABARAP), doing so with a Kd of ~5.1 μM (Fig. 1b), a finding clearly suggesting that some other region of the TM3–TM4 ICD that can interact with GABARAP
Our results showed that GST-GABARAP and GST-GABARAPL1 could bind to GFP-γ2-GABAAR full length (FL) expressed in HEK-293 cells, whereas no binding was detected for GST-LC3A or a GSTGABARAP L50A variant (Fig. 1f, see details below for the variant)
Summary
GABAA receptors (GABAARs) are the primary fast inhibitory ion channels in the central nervous system. We demonstrate that GABARAP functions to stabilize GABAARs via promoting its trafficking pathway instead of blocking receptor endocytosis. Our results reveal that GABARAP/GABARAPL1, but not other members of the Atg[8] family proteins, regulates synaptic localization of GABAARs via modulating the trafficking of the receptor. Previous studies have reported that GABARAP can directly bind to the TM3–TM4 intracellular loop of GABAA receptor γ2 subunit (γ2-GABAAR) with low affinity[18,19,20], and the co-expression of GABARAP and GABAARs in COS7 cells, quail fibroblasts, oocytes, and cultured hippocampal neurons have been shown to increase the surface levels of GABAARs21–23. A previously unrecognized fragment of the TM3–TM4 intracellular loop of γ2-GABAAR (γ2-GIM) binds to GABARAP/GABARAPL1, but not other Atg[8] family members. The γ2-GIM binds to clathrin adaptor protein AP2 with an affinity higher a b
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