Structural investigation of GluA3 AMPAR subunit reveals a unique architecture with novel allosteric coupling between the ligand-binding domain and N-terminal domain.
AMPA receptors (AMPARs) mediate the majority of excitatory neurotransmission in the brain. These ion channel tetramers are composed of four distinct subunits, named GluA1 to GluA4. Now Ingo Greger’s group, in the LMB’s Neurobiology Division, have unravelled the unique architecture of the GluA3 homomeric AMPA receptor, a subtype strongly associated with neurological disease.
First author Aditya Pokharna, a postdoctoral researcher in Ingo’s group, used electron cryo-microscopy (cryo-EM) to determine the structures of GluA3. This revealed that GluA3 adopts a structural organisation that substantially diverges from all the other AMPARs. This novel architecture is defined by a stable and previously unseen coupling between the extracellular ligand-binding domain (LBD) and N-terminal domain (NTD) tiers, creating an allosteric conduit between the areas responsible for synaptic modulation and channel gating respectively. Cryo-EM also reveals that the coupling between the two domains (NTD-LBD) is not a transient artifact but a persistent feature across the receptor’s gating cycle.
The lynchpin of this entire structure is a unique molecular feature within the GluA3 NTD: a stacking interaction between the side chains of two arginine residues (Arg163) in the NTD dimer interface. This interaction, which is unique to GluA3, traps the NTD dimer in an atypical flat conformation. It is this specific conformation that enables the NTD to make close contacts with the LBD layer. James Krieger, previously a PhD student in the Greger lab, conducted molecular dynamics (MD) simulations to validate the stability of this interface and to further delineate the conformational landscape of the receptor.
To shed light on the operational principles of GluA3 AMPA receptors, group members Josip Ivica and Imogen Stockwell functionally characterised them using electrophysiology from mammalian cells and neuronal synapses. Furthermore, targeted mutation to disrupt the residues on either interface is shown to alter the kinetics and biogenesis of the receptor complex in electrophysiology and flow cytometry assays. Interestingly, mutation of the stacking Arg163 residue substantially boosted synaptic transmission.
The team of researchers also elucidated the structural differences that dictate the cellular fate of two major GluA3 vertebrate isoforms. The GluA3-G439 (glycine 439) LBD variant is efficiently transported to the cell surface, while the mammalian-specific GluA3-R439 (arginine 439) form is largely confined to the endoplasmic reticulum when expressed alone. They revealed that the GluA3-R isoform is structurally unstable, particularly within its LBD: the LBD dimers in GluA3-R readily separate into mobile monomers, a property not observed in the stable GluA3-G. This inherent instability of the homomeric GluA3-R receptor signals for its retention within the endoplasmic reticulum and serves as a quality control mechanism, ensuring that more stable heteromeric receptor assemblies are formed before they are permitted to move to the synapse.
Despite its clinical relevance, the architectural principles governing GluA3-containing receptors had largely remained unknown. It is encoded by the gene GRIA3, located on the X-chromosome, and mutations in this gene are linked to a wide spectrum of severe neurodevelopmental and psychiatric disorders, including epilepsy, intellectual disability, aggression, autism and schizophrenia. By identifying at least three previously unknown, subtype-selective druggable hotspots in the GluA3 AMPAR, this study points to exciting new avenues for rational drug design.
This work was funded by UKRI MRC, the Wellcome Trust, Marie Skłodowska-Curie (see below) and the Spanish Research Council.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 101024130.
Further references
Architecture, dynamics and biogenesis of GluA3 AMPA glutamate receptors. Pokharna, A., Stockwell, I., Ivica, I., Singh, B., Schwab, J., Vega-Gutiérrez, C., Herguedas , B., Cais, O., Krieger, JM., Greger, I.H. Nature
Ingo’s group page
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