Control of AMPA Receptor Activity by the Extracellular Loops of Auxiliary Proteins

Abstract

The ligand gated AMPA type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission throughout the mammalian brain where they play a central role in synaptic plasticity and cognition. At the post-synapses, AMPA receptors form complexes with auxiliary subunits, including the transmembrane AMPA receptor regulating proteins (TARPs) that are known to increase receptor trafficking and modulate AMPAR properties. However, the mechanism for AMPAR gating modulation by TARPs remains poorly understood. Here, we sought to identify those sites in TARPs γ2 and γ8 responsible for modulation of channel gating. Therefore, we built structural models of TARP-AMPA receptor complexes for TARPs γ2 and γ8, combining recent structural studies and de novo structure predictions. These models, combined with peptide binding assays, provide evidence for multiple interactions between GluA2 and the variable extracellular loops of both TARPs. Substitutions and deletions of these loops had surprisingly rich effects on the kinetics of glutamate-activated currents, without any effect on assembly. Critically, substitutions in the linker domains of GluA2 completely removed any effect of γ2 on receptor kinetics, indicating a dominant role for this previously overlooked site proximal to the AMPA receptor channel gate. Likewise, by altering the two interacting loops of γ2 and γ8, we could entirely remove all allosteric modulation of GluA2, without affecting formation of AMPA receptor-TARP complexes. These findings indicate a complex set of modulatory interactions, and provide tools for future studies of TARP action in the brain.