The kainate receptor (KAR), a subtype of glutamate receptor, mediates excitatory synaptic responses at a subset of glutamatergic synapses. However, the molecular mechanisms underlying the trafficking of its different subunits are poorly understood. Here we use the CA1 hippocampal pyramidal cell, which lacks KAR-mediated synaptic currents, as a null background to determine the minimal requirements for the extrasynaptic and synaptic expression of the GluK2 subunit. We find that the GluK2 receptor itself, in contrast to GluK1, traffics to the neuronal surface and synapse efficiently and the auxiliary subunits Neto1 and Neto2 caused no further enhancement of these two trafficking processes. However, the regulation of GluK2 biophysical properties by Neto proteins is the same as that of GluK1. We further determine that it is the amino-terminal domains (ATDs) of GluK1 and GluK2 that control the strikingly different trafficking properties between these two receptors. Moreover, the ATDs are critical for synaptic expression of heteromeric receptors at mossy fiber-CA3 synapses and also mediate the differential dependence on Neto proteins for surface and synaptic trafficking of GluK1 and GluK2. These results highlight the fundamental differences between the two major KAR subunits and their interplay with Neto auxiliary proteins.

Long-term potentiation (LTP) is a persistent strengthening of synaptic transmission in the brain and is arguably the most compelling cellular and molecular model for learning and memory. Previous work found that both AMPA receptors and exogenously expressed kainate receptors are equally capable of expressing LTP, despite their limited homology and their association with distinct auxiliary subunits, indicating that LTP is far more promiscuous than previously thought. What might these two subtypes of glutamate receptor have in common? Using a single-cell molecular replacement strategy, we demonstrate that the AMPA receptor auxiliary subunit TARP γ-8, via its PDZ-binding motif, is indispensable for both basal synaptic transmission and LTP. Remarkably, kainate receptors and their auxiliary subunits Neto proteins share the same requirement of PDZ-binding domains for synaptic trafficking and LTP. Together, these results suggest that a minimal postsynaptic requirement for LTP is the PDZ binding of glutamate receptors/auxiliary subunits to PSD scaffolding proteins.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and predominantly assemble as heterotetramers in the brain. Recently, the crystal structures of homotetrameric GluA2 demonstrated that AMPARs are assembled with two pairs of conformationally distinct subunits, in a dimer of dimers formation. However, the structure of heteromeric AMPARs remains unclear. Guided by the GluA2 structure, we performed cysteine mutant cross-linking experiments in full-length GluA1/A2, aiming to draw the heteromeric AMPAR architecture. We found that the amino-terminal domains determine the first level of heterodimer formation. When the dimers further assemble into tetramers, GluA1 and GluA2 subunits have preferred positions, possessing a 1-2-1-2 spatial assembly. By swapping the critical sequences, we surprisingly found that the spatial assembly pattern is controlled by the excisable signal peptides. Replacements with an unrelated GluK2 signal peptide demonstrated that GluA1 signal peptide plays a critical role in determining the spatial priority. Our study thus uncovers the spatial assembly of an important type of glutamate receptors in the brain and reveals a novel function of signal peptides.

The X-linked GRIA3 gene encodes the GLUA3 subunit of AMPA-type glutamate receptors. Pathogenic variants in this gene were previously reported in neurodevelopmental diseases, mostly in male patients but rarely in females. Here we report a de novo pathogenic missense variant in GRIA3 (c.1979G>C; p. R660T) identified in a 1-year-old female patient with severe epilepsy and global developmental delay. When exogenously expressed in human embryonic kidney (HEK) cells, GLUA3_R660T showed slower desensitization and deactivation kinetics compared to wildtype (wt) GLUA3 receptors. Substantial non-desensitized currents were observed with the mutant but not for wt GLUA3 with prolonged exposure to glutamate. When co-expressed with GLUA2, the decay kinetics were similarly slowed in GLUA2/A3_R660T with non-desensitized steady state currents. In cultured cerebellar granule neurons, miniature excitatory postsynaptic currents (mEPSCs) were significantly slower in R660T transfected cells than those expressing wt GLUA3. When overexpressed in hippocampal CA1 neurons by in utero electroporation, the evoked EPSCs and mEPSCs were slower in neurons expressing R660T mutant compared to those expressing wt GLUA3. Therefore our study provides functional evidence that a gain of function (GoF) variant in GRIA3 may cause epileptic encephalopathy and global developmental delay in a female subject by enhancing synaptic transmission.