Architecture of fully occupied GluA2 AMPA receptor–TARP complex elucidated by cryo-EM

Abstract

Fast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPA-receptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates the kinetics of receptor gating and pharmacology, as well as trafficking. Here we report the cryo-EM structure of the homomeric GluA2 AMPA receptor saturated with TARP γ2 subunits, showing how the TARPs are arranged with four-fold symmetry around the ion channel domain, making extensive interactions with the M1, M2 and M4 TM helices. Poised like partially opened ‘hands’ underneath the two-fold symmetric ligand binding domain (LBD) ‘clamshells’, one pair of TARPs are juxtaposed near the LBD dimer interface, while the other pair is near the LBD dimer-dimer interface. The extracellular ‘domains’ of TARP are positioned to not only modulate LBD ‘clamshell’ closure, but also to affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane (TM) domains buttress the ion channel pore.

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

“…The elimination of modulation by nullifying L2 of g2, or by mutating residues in the LBD-TMD linkers of GluA2, strongly implicates this site as a pivotal interaction underlying modulation. Putative electrostatic interactions posited from structural studies require a large conformational change (between 13 Å and 25 Å depending on the TARP's position in the complex; measured between C-alpha atoms from GluA2 K699 and g2 D92 in cryo-EM complexes 5kbu and 5kk2, respectively) (Twomey et al, 2016;Zhao et al, 2016). A key point here is that these interactions are secondary to those involving L2 at the AMPAR linkers.…”

“…A key point here is that these interactions are secondary to those involving L2 at the AMPAR linkers. These interactions should occur readily for each auxiliary protein subunit, allowing a maximal 4:4 stoichiometry with minimal conformational change for g2 ( Figure 8A) (Zhao et al, 2016). For other auxiliary proteins, for example g8, the stoichiometry of the L2-linker interaction would vary with the number of associated TARPs, but will not be limited by position of the TARP within the complex ( Figure 8B).…”

“…Crystal structures of Claudins, proteins with close homology to TARPs, enabled a more refined view, defining a folded extracellular 'cap' (Suzuki et al, 2014;Saitoh et al, 2015;Shinoda et al, 2016) that substantially limits the sections of the extracellular portion of TARPs that are able to interact with the AMPA receptor, and therefore the likely range of these interactions. More recently, cryo-EM/single particle analysis of GluA2-TARP complexes allowed unambiguous positioning of TARPs at the periphery of the GluA2 pore, and partially resolved the extracellular domains of TARPs (Twomey et al, 2016;Zhao et al, 2016). The major sequence and structural differences between Claudin and TARP proteins, and between TARPs with different modulatory effects, are found in the variable extracellular loops between b1 and b2 (Loop 1), and between TM3 and b5 (Loop 2, Figure 1A).…”

“…However, in accordance with our hypothesis the majority of the L1 of both g2 and g8 contain hits (dark spots within the purple boxes, Figure 1C) in the peptide mapping array, indicating direct interaction with the receptor LBD ( Figure 1C and D, Figure 1-figure supplement 1C), albeit in conditions lacking the usual steric constraints of the complex. In the recent cryo-EM structures of the GluA2-TARP complex a possible interaction between a conserved negatively charged region (negative patch, NP) located on the TARP b4-TM2 loop and the KGK motif in the lower lobe of the GluA2 LBD was predicted (Zhao et al, 2016;Twomey et al, 2016). Thus we also tested for this potential interaction in the peptide mapping array but found no hits.…”

“…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). Particularly, we expected from our structural models and other available structural data (Twomey et al, 2016;Zhao et al, 2016) that L2 should interact with the S1-M1 linker and the S2-M4 linker in the AMPA receptor. Previous work has shown the importance of these linkers in glutamate receptor gating (Balannik et al, 2005;Schmid et al, 2007;Talukder et al, 2010).…”