The Three-dimensional Structure of an Ionotropic Glutamate Receptor Reveals a Dimer-of-dimers Assembly

The ionotropic glutamate receptors (iGluRs) represent a major family of ion channels whose quaternary structure has not yet been defined. Here, we present the three-dimensional structure of a fully assembled iGluR, determined at ∼20 Å resolution by electron microscopy. Analysis of negatively stained single-particle images reveals the presence of 2-fold, but not 4-fold, symmetry for these tetrameric channels, providing the first direct structural evidence for a dimer-of-dimers assembly. The receptor appears elongated, measuring ∼170 Å×140 Å×110 Å, with the 2-fold symmetry centered on its longitudinal axis. The overall molecular shape and symmetry suggest an orientation relative to the membrane and permit the identification of a putative transmembrane domain. Internal cavities located along the longitudinal axis may represent components of the ion conduction pathway.

Introduction

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its cognate receptors include ligand-gated ion channels that play important roles in neuronal development, in synaptic plasticity, and in a variety of neurological disorders.¹ Binding of agonist to an ionotropic glutamate receptor (iGluR) triggers the opening of an ion pore, allowing the flow of selected cations across the membrane. In the continuing presence of agonist, the receptor typically desensitizes, closing the channel. A subfamily of iGluRs exhibits high affinity for the synthetic agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and is responsible for most fast excitatory synaptic signaling in the brain. However, despite their physiological importance, the subunit organization and symmetry of the iGluRs has remained elusive.

Like other iGluRs, AMPA receptors are assembled as homo- or heterotetramers of subunits. Referred to as GluRA–GluRD (or GluR1–GluR4), these subunits are themselves modular.² They consist of two extracellular domains, each with homology to the bilobate bacterial periplasmic binding proteins.³ A membrane-distal N-terminal domain (NTD) is coupled to a membrane-proximal ligand-binding domain (LBD) that contains the agonist-binding site. The crystallographic structure of the LBD has been determined, revealing a bilobate structure that undergoes an agonist-induced cleft closure, the magnitude of which is correlated with the extent of channel activation.⁴ The LBD is connected in turn to the transmembrane domains, which exhibit partial homology to the primitive potassium channels.⁵ The C terminus is intracellular and mediates protein–protein interactions important in iGluR trafficking and localization.⁶

The following findings suggest that the component domains associate via dimeric interactions and that the receptors may not assemble as 4-fold symmetric tetramers. First of all, the LBD dimerizes weakly across an interface that appears to regulate channel desensitization.7, 8 The LBD dimerization interaction has been visualized crystallographically: the domains associate via their membrane-distal lobes in a “back-to-back” fashion, with their membrane-attachment sites pointing in the same direction and their binding clefts facing away from each other.⁴ In addition, Ayalon & Stern-Bach⁹ used subunit chimeras to develop a model of sequential dimerization for receptor assembly, in which NTD compatibility is required for an initial obligate subunit pairing, while the LBDs and transmembrane domains stabilize a subsequent dimer–dimer association. This is consistent with the observation that the NTD dimerizes more strongly in solution than the LBD,¹⁰ although GluRD subunits lacking the NTD can still assemble to form functional channels.¹¹ Further indirect support for a 2-fold symmetrical assembly is provided by the characteristics of channels formed by tandem subunits¹² and by scanning cysteine mutagenesis data.¹³ The latter data also suggest that the 2-fold symmetry of the iGluRs may apply not only to the soluble extracellular domains, but also to at least that part of the transmembrane domains located in the extracellular leaflet of the plasma membrane, despite sequence similarity to the 4-fold symmetrical K⁺-channel family.5, 14

Based on these biochemical and structural results, a working model for the assembly, activation and desensitization of the iGluRs has emerged.² Nevertheless, to date, evidence for the shape and symmetry of fully assembled glutamate receptor ion channels remains indirect. Based on our ability to purify tetrameric GluRB homomers,¹⁵ we present here the first three-dimensional (3D) structure of an AMPA receptor, obtained by electron microscopy of negatively stained specimens and single-particle image analysis. The molecular envelope provides direct structural evidence for the overall 2-fold symmetry of the channel and suggests a probable orientation relative to the lipid bilayer.