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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 Annual Report of the Division of Intramural Research

Structural Biology of Glutamate Receptor Ion Channels

Mark Mayer
  • Mark L. Mayer, PhD, Chief, Laboratory of Cellular and Molecular Neurophysiology
  • Carla Glasser, PhD, Technical Specialist
  • Sagar Chittori, PhD, Visiting Fellow
  • Poorva Dharkar, PhD, Visiting Fellow
  • Richard Grey, BSc, Postbaccalaureate Fellow
  • Austin Zimmet, BSc, Postbaccalaureate Fellow

Ionotropic glutamate receptors (iGluRs) are membrane proteins that act as molecular pores permeable to sodium and calcium ions. iGluRs mediate rapid signal transmission at the majority of excitatory synapses in the mammalian nervous system, converting the pre-synaptic action potential–triggered release of glutamate into a depolarizing post-synaptic potential. The seven iGluR gene families in humans encode 18 subunits, which assemble to form three major functional families named after the ligands that were first used to identify iGluR subtypes in the late 1970s: AMPA, kainate, and NMDA. Given the essential role of iGluRs in normal brain function and development, and mounting evidence that dysfunction of iGluR activity mediates several neurological and psychiatric diseases as well as damage during stroke, we devote substantial effort to analyzing iGluR function at the molecular level. Atomic-resolution structures solved by protein crystallization and X-ray diffraction or by single-molecule cryo-EM provide a framework for designing electro-physiological and biochemical experiments aimed at defining the mechanisms underlying ligand recognition, the gating of ion channel activity, and the action of allosteric modulators. Information derived from these experiments will permit the development of subtype-selective antagonists and allosteric modulators with novel therapeutic applications and reveal the inner workings of a complicated protein machine that plays a critical role in brain function.

Key issues in the field include obtaining structures for iGluRs trapped in their three major conformational states, i.e., the resting, activated, and desensitized states, and obtaining insight into the energy landscapes connecting the states. Also of interest are the evolutionary relationships connecting iGluRs in different species and how structurally related chemosensors bind to a wide range of small molecules.

Structural studies on full-length AMPA and kainate receptors

Our initial studies on the glutamate receptor GluK2 were limited by poor partitioning of GluK2 into the holes of holey carbon support grids, a common problem that frequently limits structural determination by single-particle cryo-electron microscopy (cryo-EM). This was overcome by depositing, on gold-coated carbon grids, a self-assembled monolayer whose surface properties were controlled by chemical modification. The procedure allowed partitioning of ionotropic glutamate receptors into the holes, thereby enabling higher resolution structural analysis than by using single-particle cryo-EM methods. Using the approach, in collaboration with the Subramaniam lab, we solved a series of structures for iGluRs trapped in different conformational states, using high-affinity ligands and allosteric modulators to determine how glutamate receptor ion channels accommodate the structural changes necessary for activation and desensitization. For the AMPA receptor GluA2, we solved structures in an antagonist-bound resting state, in a glutamate-bound active state trapped with the allosteric modulator LY451646, and in the desensitized state trapped by the high-affinity full-agonist quisqualic acid. Comparison of the closed- and active-state density maps reveals ligand-binding domain (LBD) 'clamshell' closure, as seen for isolated LBD dimers, that produces about 7 Å vertical contraction of the ATD-LBD assembly, measured as a downward movement at the top of the ATD (amino-terminal domain) tetramer, as well as unanticipated movements in the LBD, in which the dimer pairs rotate about an axis offset from the local axis of two-fold symmetry. This generates a cork screw–like motion, in which the four LBDs expand and likely unwind the iris of the M3 helix (transmembrane inner helix)–bundle closed state.

Analysis of cryo-electron microscopic images for the GluA2–desensitized state revealed evidence of substantial conformational heterogeneity at the ATD layer, precluding determination of a single desensitized-state 3D structure. Three-dimensional classification of the images enabled separation of three dominant classes at nominal resolutions of 21 Å, 23 Å, and 26 Å, with variable degrees of displacement between ATD dimers compared with the closed and active states. In all three classes, the LBD layer separates into four lobes of density, with different degrees of separation between the proximal and distal LBD subunits, strikingly different from the 'dimer-of-dimers' structure found in the closed and active states, but reminiscent of the GluK2 structure determined by tomography.

The GluK2–desensitized state was solved to a resolution of 7.6 Å and was adequate to unambiguously show that, in the desensitized state, the ion channel adopts a closed conformation in which the M3 helices form a crossed-bundle assembly with the pre-M1 helices (transmembrane outer helix) wrapped around the outside of the channel, similar to that seen for the antagonist-bound closed state, and revealed for the first time how the LBDs rearrange to permit the ion channel to close, even though the individual subunits retain a glutamate-bound closed-cleft active conformation. Desensitization occurs because, in the LBD assembly, the distal subunits swing clockwise by 125° in the horizontal plane, while the proximal subunits rotate by 13°. In the vertical plane, the distal and proximal subunits tilt 11° and 6° away from the global axis of symmetry. As a result of these movements, in the GluK2–desensitized state the LBD layer resembles an inverted pyramid, in which the four subunits are arranged with quasi four-fold symmetry, strikingly different from the two-fold symmetric dimer-of-dimers assembly in the active state. Overall, the results provide a detailed glimpse into the overall gating cycle of glutamate receptors, an evaluation of the similarities and differences in conformational changes observed in AMPA– and kainate-receptor families, and a molecular mechanism for the marked LBD movements that occur during the receptor gating cycle.

Drosophila larval neuromuscular junction glutamate receptors

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. In a collaboration with Mihaela Serpe, we found that the auxiliary subunit Drosophila Neto functions as an essential component required for the function of NMJ glutamate receptors in heterologous expression systems, in accord with the results of genetic analysis that revealed paralysis in Neto knockouts. In combination with a crystallographic analysis of the GluRIIB ligand-binding domain, we used this system to characterize the subunit dependence of assembly, channel block, and ligand selectivity for Drosophila NMJ glutamate receptors.

We found that Neto weakly modulates, but is not required for, cell-surface expression of Drosophila iGluRs. Instead Neto profoundly increases receptor activation by glutamate. However, even with Neto, efficient receptor cell-surface expression and function requires coexpression of four different iGluR subunits, such that responses for GluRIIA/E and GluRIIA/C/E were on average still only 2–3% of the amplitude of those recorded for GluRIIA/C/D/E. Current-voltage plots for glutamate responses revealed pronounced biphasic rectification, as a result of channel block by cytoplasmic polyamines, consistent with the absence of mRNA editing for Drosophila iGluRs and the presence of a glutamine residue at the Q/R site of the pore loop. Argiotoxin (ATX) produced block of responses to glutamate at −60 mV for both GluRIIA/C/D/E and GluRIIB/C/D/E but with much faster recovery from block for GluRIIB/C/D/E. We then tested for activation by AMPA, kainate, and N-methyl-¿-aspartate (NMDA), the canonical ligands used to classify vertebrate iGluRs, and found that none of these produced functional responses, with only glutamate and quisqualate acting as agonists. The structural basis for this was revealed by the crystal structure of the GluRIIB ligand-binding domain, which revealed glutamate trapped in a cavity of volume 208 Å3 together with three water molecules. Within domain 1 of the GluRIIB LBD structure, in the loop between β-strand 7 and α-helix D, the side chain of Asp509 forms a hydrogen bond with the hydroxyl group of Tyr481, a conserved aromatic residue that caps the entrance to the ligand-binding cavity, sealing it from extracellular solvent. Stacked above Tyr481, the side chain of Arg429 forms a cation pi interaction with the aromatic ring, further stabilizing the conformation of Tyr481. Amino-acid sequence alignments reveal that Asp509 is conserved in all Drosophila NMJ iGluRs, while in all vertebrate AMPA and kainate receptor subunits there is a proline at this position; likewise cation pi stacking by Arg429 is unique to GluRIIA, GluRIIB, and GluRIIC, because vertebrate AMPA and kainate receptor subunits have an isoleucine residue at this position. Consequently, AMPA, as a result of the different conformation of the isoxazazole group, is unable to bind to GluRIIB because the ligand's 5-methyl group makes steric clashes with Asp509 and Asn736. Likewise, although the ligand α-carboxyl, α-amino and γ-carboxyl groups of kainate are isosteric with those of glutamate, the isopropenyl group makes steric clashes with the Asp509 and Tyr481 side chains, thus explaining the unique ligand-binding properties of Drosophila larval neuromuscular junction iGluRs.

Structural studies on ctenophore glutamate receptors

Recent genome projects for ctenophores (commonly known as comb jellies) revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi (ML) and Pleurobrachia bachei (Pb), which are among our earliest metazoan ancestors, perhaps evolving even before sponges and placazoans. Sequence alignments and phylogenetic analysis show that ctenophore iGluRs form a distinct clade from the well characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) at resolutions of 1.21 and 1.5 Å reveal endogenous glycine in the binding pocket, while ligand-binding assays show that glycine binds with nM affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge present only in subunits that bind to glycine, but also a conserved disulfide in loop 1 of the LBD that is found in vertebrate NMDA but not AMPA or kainate receptors. In electrophysiological experiments, we found that ML032222a forms homomeric glycine-activated ion channels, while ML05909a forms functional homomeric receptors that are activated by glutamate and for which glycine acts as a weak partial agonist. Because the affinity of ML05909a for glycine is greater than that for glutamate, and because the efficacy of glycine is so low, glycine acts as a functional glutamate antagonist, analogous to the partial agonist action of HA-966 on the GluN1 subunit of vertebrate NMDA receptors.

Could ML032222a, PbiGluR3, and related ctenophore iGluRs be relatives of NMDA receptor subunits that bind to glycine? Was binding of glycine a common feature of primitive iGluRs which subsequently evolved to bind to glutamate with high affinity? A surprising structural feature in all ctenophore iGluRs, which is revealed by our structural analysis, is a conserved disulfide bond in loop 1 that is found only in NMDA receptor subunits, including those from the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, but not in AMPA or kainate receptors. Did the ctenophore iGluRs acquire this signature after splitting from the last common ancestor of other animal families, or was this feature present in a primordial glutamate receptor that subsequently evolved to give rise to different iGluR clades? Our results highlight the difficulty of classifying the ligand-binding and functional properties of newly discovered iGluRs identified by genome sequencing projects, and suggest that attempts to classify iGluRs from invertebrate species into classes using the scheme developed for vertebrate AMPA, kainate, and NMDA receptors is not reliable and should be avoided.


  1. Alberstein R, Grey R, Zimmet A, Simmons DK, Mayer ML. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes. Proc Natl Acad Sci USA 2015; 112(44):E6048-57.
  2. Han TH, Dharkar P, Mayer ML, Serpe M. Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors. Proc Natl Acad Sci USA 2015; 112:6182-6187.
  3. Meyerson JR, Kumar J, Chittori S, Rao P, Pierson J, Bartesaghi A, Mayer ML, Subramaniam S. Structural mechanism of glutamate receptor activation and desensitization. Nature 2014; 514:328-334.
  4. Schauder DM, Kuybeda O, Zhang J, Klymko K, Bartesaghi A, Borgnia MJ, Mayer ML, Subramaniam S. Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc Natl Acad Sci USA 2013; 110:5921-5926.
  5. Yao Y, Belcher J, Berger AJ, Mayer ML, Lau AY. Conformational analysis of NMDA receptor GluN1, GluN2, and GluN3 ligand-binding domains reveals subtype-specific characteristics. Structure 2013; 21:1788-1799.


  • Albert Lau, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
  • Chi-Hon Lee, MD, PhD, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
  • Peter Schuck, PhD, Laboratory of Cellular Imaging and Macromolecular Biophysics, NIBIB, Bethesda, MD
  • Mihaela Serpe, PhD, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
  • Sriram Subramaniam, PhD, Laboratory of Cell Biology, Center for Cancer Research, NCI, Bethesda, MD
  • Joy Zhao, PhD, Laboratory of Cellular Imaging and Macromolecular Biophysics, NIBIB, Bethesda, MD

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