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.

中文翻译：

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GABA A 型受体中 α2 亚基 Ser-359 的磷酸化下调了它们在抑制性突触处的密度。

GABA A 型受体 (GABAARs) 介导快速突触抑制，并被贩卖到功能多样的突触。然而，调节这些受体突触靶向的精确分子机制尚不清楚。尽管先前已表明 GABAAR 的 α4、β 和 γ 亚基中的磷酸化事件控制其功能和运输，但尚未证明其他亚基的磷酸化。在这里，我们表明 GABAARs 的 α2 亚基在 Ser-359 处被磷酸化，并能够动态调节 GABAAR 与支架蛋白 gephyrin 和 collybistin 的结合。我们最初通过 MS 分析确定了 Ser-359 磷酸化，并且其他实验表明它受 cAMP 依赖性蛋白激酶 (PKA) 和蛋白磷酸酶 1 (PP1) 和/或 PP2A 的活性调节。基于 GST 的 pulldowns 和共免疫沉淀实验表明 gephyrin 和 collybistin 优先结合 WT 和 S359A phosphonull 变体，但不结合 S359D 拟磷酸化变体。此外，α2 S359D 变体结合 collybistin 和 gephyrin 的能力降低降低了含有突触 α2 的 GABAAR 簇的密度，并导致轴突起始段缺乏 α2 富集。这些结果表明，PKA 介导的 α2 亚基磷酸化和 PP1/PP2A 依赖性去磷酸化在抑制性突触处 GABAAR 积累的动态调节中发挥作用，从而调节突触抑制的强度。MS 数据已存入 ProteomeXchange，数据集标识符为 PXD019597。