Phosphorylation on Ser-359 of the α2 subunit in GABA type A receptors down-regulates their density at inhibitory synapses

Yasuko Nakamura,Danielle H Morrow,Anna J Nathanson,Jeremy M Henley,Kevin A Wilkinson,Stephen J Moss

doi:10.1074/jbc.ra120.014303

Yasuko Nakamura, Danielle H Morrow + Show 4 more

Open Access

https://doi.org/10.1074/jbc.ra120.014303

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Abstract

GABA type A receptors (GABAARs) mediate fast synaptic inhibition and are trafficked to functionally diverse synapses. However, the precise molecular mechanisms that regulate the synaptic targeting of these receptors are unclear. Whereas it has been previously shown that phosphorylation events in α4, β, and γ subunits of GABAARs govern their function and trafficking, phosphorylation of other subunits has not yet been demonstrated. Here, we show that the α2 subunit of GABAARs is phosphorylated at Ser-359 and enables dynamic regulation of GABAAR binding to the scaffolding proteins gephyrin and collybistin. We initially identified Ser-359 phosphorylation by MS analysis, and additional experiments revealed that it is regulated by the activities of cAMP-dependent protein kinase (PKA) and the protein phosphatase 1 (PP1) and/or PP2A. GST-based pulldowns and coimmunoprecipitation experiments demonstrate preferential binding of both gephyrin and collybistin to WT and an S359A phosphonull variant, but not to an S359D phosphomimetic variant. Furthermore, the decreased capacity of the α2 S359D variant to bind collybistin and gephyrin decreased the density of synaptic α2-containing GABAAR clusters and caused an absence of α2 enrichment in the axon initial segment. These results suggest that PKA-mediated phosphorylation and PP1/PP2A-dependent dephosphorylation of the α2 subunit play a role in the dynamic regulation of GABAAR accumulation at inhibitory synapses, thereby regulating the strength of synaptic inhibition. The MS data have been deposited to ProteomeXchange, with the data set identifier PXD019597.

Highlights

GABA type A receptors (GABAARs) mediate fast synaptic inhibition, the efficacy of which is determined by the number of inhibitory synapses [1, 2]
Number of synaptic a2 clusters in dendrites and an absence of a2 enrichment in the axon initial segment (AIS). These results show that the phosphoregulation of a2 by protein kinase (PKA) and phosphatase 1 (PP1)/phosphatase 2A (PP2A) allows neurons to adjust the efficacy of gephyrin and collybistin binding, which plays a fundamental role in regulating the density of inhibitory synapses at dendrites and the enrichment of inhibitory synapses at the AIS
To determine the contribution of phosphorylation to this process, we examined whether the GABAAR a2 subunits were phosphorylated endogenously by utilizing a previously characterized pHluorin GABAAR a2 knock-in (pHa2) mouse that allows rapid receptor purification using GFP-Trap [22]

Summary

Introduction

GABAARs mediate fast synaptic inhibition, the efficacy of which is determined by the number of inhibitory synapses [1, 2]. These results support the MS data in the identification a novel phosphorylation site at Ser-359 in the GABAAR a2 subunit. Pulldown assays were performed utilizing GST or GST fused to the large intracellular loop of GABAAR a2 WT, S359A phosphonull, or S359D phosphomimetic mutants to investigate binding of gephyrin and collybistin.

Results

Conclusion