Architecture of the heteromeric GluA1/2 AMPA receptor in complex with the auxiliary subunit TARP γ8

INTRODUCTION Neuronal communication at excitatory synapses in the brain involves release of the neurotransmitter l-glutamate from the presynapse of one cell and its detection by postsynaptic ionotropic glutamate receptors (iGluRs) on another. A principal iGluR is the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor (AMPAR). On activation, AMPARs induce depolarization of the postsynaptic membrane to mediate rapid synaptic signaling and therefore precise information transfer at synapses. Long-lasting changes in synaptic strength can occur through recruitment to, or removal of, AMPARs from the synapse in response to particular patterns of synaptic activity. These synaptic plasticity processes, such as long-term potentiation (LTP) or long-term depression (LTD), are considered a cellular basis for learning and memory. AMPARs assemble into tetramers from four core subunits, GluA1 to GluA4, in various combinations. The GluA1/2 heteromer predominates throughout the forebrain and is selectively recruited during LTP at the intensely studied hippocampal CA3-CA1 synapse. Receptor function is further diversified by association with auxiliary subunits, such as the TARP (transmembrane AMPAR regulatory protein) family. TARP γ8, a potent AMPAR modulator and the target for recent therapeutics, is selectively enriched in the hippocampus, forming a major component of the GluA1/2 signaling complex. Within the AMPAR tetramer, the core subunits arrange in two conformationally distinct pairs, termed AC and BD, which play different roles in channel opening and are differentially modulated by TARPs. Although the BD pair has dominant control of activation in iGluRs, the rules of subunit arrangement in AMPARs are unclear. RATIONALE To elucidate the architecture and subunit organization of heteromeric AMPARs, we determined the structure of the GluA1/2 receptor in complex with TARP γ8 by means of cryo–electron microscopy (cryo-EM). Because up to four TARPs can decorate an AMPAR tetramer, yet γ8 appears to preferentially associate in a two-TARP stoichiometry, we fused γ8 to the GluA2 subunit, which, when co-expressed with GluA1, allows production of GluA1/2 associated with two γ8 auxiliary subunits. In addition, we used targeted mutagenesis in electrophysiological assays to probe subunit arrangement, gating properties, and mechanisms of TARP-specific receptor modulation. RESULTS Functional assays demonstrate preferential positioning of the GluA1 subunits to the AC positions, giving the functionally critical GluA2 (BD pair) dominant control over gating. The receptor assembly adopts an overall “Y” shape, characteristic of homomeric GluA2 structures, with the two extracellular domain layers [the N-terminal domain (NTD) and ligand-binding domain (LBD)], forming a dimer-of-dimers arrangement. The arms of the Y shape are held in place through an interface between the GluA2 NTDs, dictated by their positioning to the BD sites. The γ8 subunits, associated through intramembrane interactions, locate beneath the LBD dimer-of-dimers interface, with their distinctly long extracellular loops selectively engaging the GluA2 LBD to modulate channel gating, whereas lipid-like cryo-EM densities are observed in cavities formed between γ8 and the GluA1 TMD sector. Side chain resolution of the ion selectivity filter at the heart of the channel reveals the atomic details of the calcium-restricting “Q/R editing site,” which critically determines the properties of heteromeric AMPAR assemblies throughout the brain. CONCLUSION This structural and functional characterization of the GluA1/2 TARP γ8 complex reveals the architecture of a prominent AMPAR heteromer, offering a blueprint for deciphering signaling mechanisms of synaptic AMPARs.