The Challenge of Interpreting Glutamate-Receptor Ion-Channel Structures

Abstract

Ion channels activated by glutamate mediate excitatory synaptic transmission in the central nervous system. Similar to other ligand-gated ion channels, their gating cycle begins with transitions from a ligand-free closed state to glutamate-bound active and desensitized states. In an attempt to reveal the molecular mechanisms underlying gating, numerous structures for glutamate receptors have been solved in complexes with agonists, antagonists, allosteric modulators, and auxiliary proteins. The embarrassingly rich library of structures emerging from this work reveals very dynamic molecules with a more complex conformational spectrum than anticipated from functional studies. Unanticipated conformations solved for complexes with competitive antagonists and a lack of understanding of the structural basis for ion channel subconductance states further highlight challenges that have yet to be addressed.

Figure 1

Domain organization and gating in iGluR tetramers. (A) Simplified state diagram for iGluR gating shows transitions between apo (R), agonist-bound (RA), open (O), and desensitized (D) states. (B) Minimal NMDA receptor state diagram showing that binding of both glutamate and glycine is required to open the channel; desensitized states were omitted from this model. (C) State diagram for an iGluR tetramer during sequential binding of four molecules of glutamate with transitions between apo (R), glutamate-bound (R1–R4), open (O1–O4), and desensitized (D1–D4) states. (D) Atomic coordinates from a GluK2 antagonist-bound resting-state structure, for which three subunits are drawn in transparent gray shading, with the fourth subunit colored using rainbow shading from the N-terminus (blue) to C-terminus (red), highlights assembly of both the ATD and LBD as a dimer or dimers, and connection of the ATD to LBD and LBD to TMD by linker peptides. To see this figure in color, go online.

Figure 2

Cryo-EM structures of agonist and antagonist bound iGluRs. (A) Side views of LBD dimer assemblies from full-length (CTD deleted) AMPA-receptor GluA2 cryo-EM structures for the glutamate complex (left; PDB: 4UQ6) and the competitive antagonist ZK 200775 complex (right; PDB: 4UQJ). (B) The upper structures show corresponding views for the NMDA-receptor GluN1/GluN2B LBD heterodimer assembly with glycine and glutamate (left; PDB: 5IOU) and the competitive antagonist DCKA/AP5 complex (right; PDB: 5IPS); the lower structures show the GluN1/GluN2 LBD dimer-of-dimers assembly viewed from the top, after rotation by 90°, illustrating dissociation LBD dimers for the DCKA/AP5 complex. In all structures, one subunit in the dimer assembly is colored blue (GluN1 and GluA2) and the second subunit orange (GluN2B and GluA2), with α-helices D and J shown in darker shading; dashed lines connect helices D and J within and between dimer assemblies. To see this figure in color, go online.

Figure 3

Vectors between CA atoms at the N-terminus of α-helix E for the AC- and BD-subunit LBDs illustrate how expansion that occurs in the open state is due to much larger movements of the BD subunits compared to prior x-ray and cryo-EM active-state structures of GluA2 agonist complexes stabilized by allosteric modulators (Mod) that block desensitization.