Structure and Dynamics of AMPA Receptor GluA2 in Resting, Pre-Open, and Desensitized States

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/ YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double-and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.T he α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (iGluRs) reside in postsynaptic membranes in the central nervous system (1). Like the other major iGluR subtypes, the N-methyl-Daspartate receptors (NMDARs) and kainate receptors, AMPARs are ligand-gated cation channels formed by hetero-or homotetrameric assembly of the GluA1 to GluA4 subunits into a membranespanning receptor with an integral ion channel. The energy from binding of the excitatory neurotransmitter glutamate at extracellular sites triggers AMPARs to passage through functional states that can include ion channel activation, deactivation, and desensitization. The timing and interplay between the necessarily distinct conformations of these states determine both basal transmission and short-term plasticity of the postsynapse (2-4).Structures of AMPARs captured in various functional states using X-ray crystallography and cryoelectron microscopy have recently become available (5-9). Overall, the structures confirm three distinct structural layers (Fig. 1). The amino-terminal domain (ATD) and the ligand-binding domain (LBD), both distal to the membrane, and the transmembrane ion channel are each formed from four copies of the respective domain from each subunit. However, a fourth region of the receptor extends from the intracellular side of the membrane. This part of the receptor includes intracellular loops between the transmembrane helices and the variable C-terminal domain (CTD), and remains entirely unresolved in the presently available AMPAR structures.The GluA2 structures and previous results from biochemical, biophysical, and crystallographic studies of isolated subunits and fu...

mentioning

confidence: 74%

Structural rearrangement of the intracellular domains during AMPA receptor activation

Zachariassen

Katchan

Jensen

et al. 2016

“…The crystal structures of homotetrameric GluA2 demonstrate that AMPARs are assembled with two pairs of conformationally distinct subunits (16)(17)(18). The different subunit conformations in the AMPAR complex further increase the assembly complexity of heteromeric AMPARs.…”

Section: Resultsmentioning

confidence: 99%

“…The X-ray crystal structure of GluA2 homomers demonstrates that the receptors have a twofold symmetry (16)(17)(18), with two pairs of conformationally distinct subunits. Viewed at the amino-terminal domain (ATD) level, the A-type conformation represents the subunits away from the symmetric axis, and the B-type conformation represents the subunits proximal to the symmetric center, suggesting that GluA2 homomers are thus arranged in an A-B-A′-B′ architecture (16)(17)(18). The structure of heteromeric NMDA-type glutamate receptor (GluN1/N2B) also shows a twofold symmetry, with GluN1 at the A positions and GluN2B at the B positions in a 1-2-1-2 (GluN1-GluN2-GluN1-GluN2) fashion (19).…”

mentioning

confidence: 99%

GluA1 signal peptide determines the spatial assembly of heteromeric AMPA receptors

Kalyanaraman

et al. 2016

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and predominantly assemble as heterotetramers in the brain. Recently, the crystal structures of homotetrameric GluA2 demonstrated that AMPARs are assembled with two pairs of conformationally distinct subunits, in a dimer of dimers formation. However, the structure of heteromeric AMPARs remains unclear. Guided by the GluA2 structure, we performed cysteine mutant cross-linking experiments in fulllength GluA1/A2, aiming to draw the heteromeric AMPAR architecture. We found that the amino-terminal domains determine the first level of heterodimer formation. When the dimers further assemble into tetramers, GluA1 and GluA2 subunits have preferred positions, possessing a 1-2-1-2 spatial assembly. By swapping the critical sequences, we surprisingly found that the spatial assembly pattern is controlled by the excisable signal peptides. Replacements with an unrelated GluK2 signal peptide demonstrated that GluA1 signal peptide plays a critical role in determining the spatial priority. Our study thus uncovers the spatial assembly of an important type of glutamate receptors in the brain and reveals a novel function of signal peptides.AMPA receptors | signal peptide | spatial assembly | stoichiometry

“…3A), a position that in vertebrate GluA2 and GluK2 AMPA and kainate receptors is converted to an arginine by RNA editing (20)(21)(22). In the M3 helix, which forms the entrance to the ion channel pore in glutamate receptors (23)(24)(25)(26), Drosophila GluRIIB has three amino acid substitutions; most notable is the replacement of asparagine by lysine at position 647 (Fig. 3A).…”

Section: Efficient Receptor Expression and Function Requires Multiplementioning

confidence: 99%

Functional reconstitution ofDrosophila melanogasterNMJ glutamate receptors

Han

Dharkar

Mayer

et al. 2015

The Drosophila larval neuromuscular junction (NMJ), at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute NMJ glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. We find that Drosophila Neto functions as an essential component required for the function of NMJ glutamate receptors, permitting analysis of glutamate receptor responses in Xenopus oocytes. In combination with a crystallographic analysis of the GluRIIB ligand binding domain, we use this system to characterize the subunit dependence of assembly, channel block, and ligand selectivity for Drosophila NMJ glutamate receptors.T he amino acid L-glutamate is the major neurotransmitter at vertebrate excitatory central synapses and at the neuromuscular junction (NMJ) of insects and crustaceans (1-3). Many vertebrate AMPA and kainate receptors, as well as GluRI from the worm Caenorhabditis elegans and AvGluR1 from the rotifer Adineta vaga, can form functional homomeric ion channels (1, 4, 5). In contrast, genetic studies suggest that assembly of Drosophila NMJ type A and type B glutamate receptors requires four subunits: either GluRIIA or GluRIIB, plus GluRIIC, GluRIID, and GluRIIE (6-9); both subtypes desensitize rapidly, with time constants of 18.8 and 2.0 ms, respectively (6). In flies, lack of GluRIIA and GluRIIB or any other single subunit induces embryonic paralysis. Furthermore, in the absence of GluRIIA and GluRIIB, or any other subunit, none of the remaining iGluR subunits cluster at nascent synapses, suggesting that recruitment and stabilization of iGluRs at synaptic sites requires heterotetramers. Despite a decade of work, the molecular basis for this unique profile has remained obscure. In part, this is because the reconstitution of functional glutamate receptors (iGluRs) in heterologous systems, which has been a powerful tool for analysis of receptor function in other species, has not been achieved for the Drosophila NMJ (10).We recently demonstrated that the synaptic distribution of Drosophila NMJ iGluRs requires Neto (Neuropillin and Tolloidlike), a protein essential for NMJ function (11). Neto belongs to a family of highly conserved auxiliary proteins that modulate the function of vertebrate kainate receptors (12) and C. elegans AMPA receptors (13,14). In these species Neto plays a minor role in delivery and synaptic targeting, and instead modulates receptor gating properties. In contrast, in flies Neto is absolutely required for clustering of iGluRs at the NMJ and lack of Neto induces embryonic paralysis (11). This finding may reflect a role for Neto in receptor assembly, surface expression, synaptic trafficking and stabilization, or modulation of iGluR gating. To distinguish among these possibilities, a recombinant expression system for...