The impact of subunit type, alternative splicing, and auxiliary proteins on AMPA receptor trafficking

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.