Couch Tyler, McCullock Tyler W, MacLean David M
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, USA.
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, USA.
J Biol Chem. 2025 Apr 30;301(6):108569. doi: 10.1016/j.jbc.2025.108569.
AMPA receptors underlie fast excitatory synaptic transmission in the mammalian nervous system and are critical for the expression of synaptic plasticity. Four genes encode the AMPA receptor subunits, each subject to RNA editing and alternative splicing at multiple positions. In addition, each tetrameric AMPA receptor can harbor up to four auxiliary proteins of which there are multiple types. Subunit type, alternative splicing, and auxiliary proteins are all known to affect AMPA receptor gating and trafficking. However, determining which factors dominate AMPA receptor trafficking requires high-throughput assessment of trafficking across multiple conditions. Here, we deploy two such methods to assess the relative contribution of AMPA receptor subunit type (GluA1 versus GluA2), alternative splicing (flip versus flop), and various transmembrane AMPA receptor regulatory proteins (TARPs) to AMPA receptor trafficking. We find that subunit type is the most important factor, with GluA2 showing a much better surface expression than GluA1, and alternative splicing plays a secondary role, with flip subunits consistently outperforming flop variants in surface expression across all conditions. Type 1 TARPs (γ2-4 and γ8) enhance surface trafficking, while Type 2 TARPs (γ5 and γ7) reduce surface expression, although we could not detect differences within each type. These data will be a helpful resource in comparing surface expression across a variety of AMPA receptor compositions. Our assays will also enable high-throughput assessment of novel disease-associated mutations, chimeras, and auxiliary and chaperone proteins.
AMPA受体是哺乳动物神经系统中快速兴奋性突触传递的基础，对突触可塑性的表达至关重要。四个基因编码AMPA受体亚基，每个亚基在多个位置都经历RNA编辑和可变剪接。此外，每个四聚体AMPA受体最多可容纳四种辅助蛋白，且辅助蛋白有多种类型。已知亚基类型、可变剪接和辅助蛋白都会影响AMPA受体的门控和转运。然而，要确定哪些因素在AMPA受体转运中起主导作用，需要在多种条件下对转运进行高通量评估。在这里，我们采用两种这样的方法来评估AMPA受体亚基类型（GluA1与GluA2）、可变剪接（翻转与翻转）以及各种跨膜AMPA受体调节蛋白（TARPs）对AMPA受体转运的相对贡献。我们发现亚基类型是最重要的因素，GluA2的表面表达比GluA1好得多，可变剪接起次要作用，在所有条件下，翻转亚基在表面表达上始终优于翻转变体。1型TARPs（γ2 - 4和γ8）增强表面转运，而2型TARPs（γ5和γ7）降低表面表达，尽管我们在每种类型内未检测到差异。这些数据将有助于比较各种AMPA受体组成的表面表达。我们的检测方法还将能够对新的疾病相关突变、嵌合体以及辅助蛋白和伴侣蛋白进行高通量评估。
