Neuronal communication occurs at specialised sites, known as synapses. Presynaptic neurotransmitter release from one neuron acts on the postsynaptic receptors of another, facilitating transfer of electrical impulses between neurons. At glutamatergic synapses, ionotropic glutamate receptors (iGluRs) respond to presynaptically released L-glutamate, the predominant excitatory neurotransmitter. Excitatory synaptic transmission is mediated primarily by AMPA-type glutamate receptors (AMPARs), and is crucial for the transfer and storage of information in the brain. The AMPAR engages in numerous protein interactions, modulating receptor trafficking, channel gating, synaptic positioning, and ultimately synaptic strength.

iGluR family members bind synaptic proteins through their N-terminal domain (NTD), influencing synaptic and neuronal circuit function. The AMPAR NTD encompasses 50% of the receptor, extending midway into the synaptic cleft, exposing it to a protein-rich environment. The strength of synaptic transmission is dependent on the AMPAR NTD, likely by positioning receptors at the synapse. This mechanism is thought to be regulated through direct protein interactions with the NTD. The identity and functional importance of these protein interactions however, are unknown. Therefore, this study aims to identify and characterise the synaptic function of AMPAR NTD-interacting proteins.

Aided by the development of a novel proximity-labelling proteomics technique, synaptic proteins enriched for the AMPAR NTD were determined. This permitted identification of transient NTD interactors, previously unresolved using classical affinity-purifications. Furthermore, this technique provides a comprehensive list of diverse synaptic cleft proteins, which may influence AMPAR function through direct or indirect mechanisms. Candidate interactors were later screened for direct interactions with iGluR NTDs by establishing a cell-based binding assay. This confirmed known iGluR NTD interactors and uncovered neuronal pentraxin-1 (Nptx1) as an AMPAR NTD-interacting protein. Facilitated by structural biology, the Nptx1 binding site was deduced using AMPAR NTD mutants. The functional consequence of this interaction was then studied at synapses onto principle neurons of the hippocampus using different electrophysiological and imaging approaches, demonstrating Nptx1 as a subunit-specific regulator of AMPAR-mediated synaptic transmission. These findings have profound implications for understanding the role of AMPAR-interacting proteins in regulating the strength of synaptic connections.