Structural Bases of Desensitization in AMPA Receptor-Auxiliary Subunit Complexes

Abstract

SUMMARY Fast excitatory neurotransmission is mediated by AMPA-subtype ionotropic glutamate receptors (AMPARs). AMPARs, localized at post-synaptic densities, are regulated by transmembrane auxiliary subunits that modulate AMPAR assembly, trafficking, gating and pharmacology. Aberrancies in AMPAR-mediated signaling are associated with numerous neurological disorders. Here, we report cryo-EM structures of an AMPAR in complex with the auxiliary subunit GSG1L in the closed and desensitized states. GSG1L favors the AMPAR desensitized state, where channel closure is facilitated by profound structural rearrangements in the AMPAR extracellular domain, with ligand-binding domain dimers losing their local two-fold rotational symmetry. Our structural and functional experiments suggest that AMPAR auxiliary subunits share a modular architecture and use a common transmembrane scaffold for distinct extracellular modules to differentially regulate AMPAR gating. By comparing the AMPAR-GSG1L complex structures, we map conformational changes accompanying AMPAR recovery from desensitization and reveal structural bases for regulation of synaptic transmission by auxiliary subunits.

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

“…Deletion of L1 from g2 and g8 approximately halved the steady state current (15 ± 2 and 15 ± 3%, n = 11 and 15, for g2 DL1 and g8 DL1, respectively; Figure 2B and D and Table 1), with a barely detectable speeding up of entry to desensitization (60 ± 5 s -1 , n = 11 and 15, for g2 DL1 and g8 DL1, respectively; Figure 2B and C and Table 1). These results suggested that L1 can influence desensitization of complexes, as shown recently for GSG1L (Twomey et al, 2017a) but the absence of a simple exchange in desensitization behavior suggested that this loop functions in concert with other modulatory elements. …”

“…Because in these experiments, slow desensitization occurs when occupancy of superactive states is low, we can quite reasonably assume that L1 adopts multiple conformations to stabilize separate functional states of the receptor, and that some functional signatures require a concerted action of both loops. Additional stabilization of desensitized states by the variable loop 1 is also likely (Twomey et al, 2017a).…”

“…From our models, a range of sites on GluA2 could interact with L1, including the KGK motif in the LBD (Twomey et al, 2017a;Dawe et al, 2016). Substitutions at L2 of g2 and g8 had profound effects on gating of TARP complexes and are well placed to interact with gating machinery ( Figure 1B and Figure 1-figure supplement 1B).…”

“…However, the C-termini of both the AMPA receptor and TARPs also appear to be involved (BenYaacov et al, 2017). Despite these insights, there is very little information about the extent to which different domains contribute to gating of complexes (Twomey et al, 2017a), and no information about the structural basis of slow modulation, superactivation (Carbone and Plested, 2016).…”

Channel opening and gating mechanism in AMPA-subtype glutamate receptors

“…We used cryo-EM to analyze the structure of the homomeric GluA2 flip splice variant AMPAR in complex with the auxiliary subunit germline-specific gene 1-like (GSG1L), which we surmised would stabilize the closed-state structure of the ion channel as it lowers the ion channel open probability compared to the transmembrane AMPAR regulatory protein (TARP)-γ2 or stargazin (STZ) 15,16 . Indeed, using a covalent fusion construct (Methods) between truncated forms of GluA2 and GSG1L, we solved structures of the digitonin-solubilized receptor complex bound to the antagonist ZK200775 (ZK) in two states, termed GluA2-GSG1L ZK-1 (4.6 Å) and GluA2-GSG1L ZK-2 (4.4 Å) (Extended Data Table 1; Extended Data Fig.…”