N‐glycosylation of the AMPA‐type glutamate receptor regulates cell surface expression and tetramer formation affecting channel function

Abstract

The AMPA-type glutamate receptor (AMPA-R) plays a primary role in principal excitatory synaptic transmission and many neuronal functions including synaptic plasticity that underlie learning and memory. N-glycosylation is one of the major post-translational modifications of membrane proteins, but its specific roles in neurons remain largely unknown. AMPA-R subunits are N-glycosylated at their extracellular domains during their biosynthesis in the lumen of the endoplasmic reticulum and Golgi system. Six N-glycosylation sites are presumed to exist in the extracellular domain of GluA1, which is a member of the AMPA-R subunits. We observed that the intracellular trafficking and cell surface expression were strongly suppressed in the GluA1 mutants lacking N-glycans at N63/N363 in HEK293T cells. Multimer analysis using Blue Native-PAGE displayed the impaired tetramer formation in the glycosylation mutants (N63S and N363S), indicating that the mis-transport was caused by impaired tetramer formation. N63S and N363S mutants were primarily degraded via the lysosomal pathway. Flag-tagged N363S GluA1, but not N63S GluA1, expressed in primary cortical neuron cultures prepared from GluA1 knockout mice was observed to localize at the cell surface. Co-expression of GluA2 partially rescued tetramer formation and the cell surface expression of N363S GluA1 but not N63S GluA1, in HEK293T cells. Electrophysiological analysis also demonstrated functional heteromers of N363S GluA1 with GluA2. These data suggest that site-specific N-glycans on GluA1 subunit regulates tetramer formation, intracellular trafficking, and cell surface expression of AMPA-R. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions

“…Among the GluA1 and GluA2 N-glycosylation site mutants, the cell surface expression levels of N63 and N363 mutants for GluA1 and the N370 mutant for GluA2 were considerably lower than WT or other N-glycosylation mutants in HEK293 cells at 24-48 h post-transfection [17,20]. Consistent with these previous reports, we detected GluA1WT and GluA1N257Q on the cell surface at 24 h post-transfection ( Figure 1).…”

“…Similarly, the inhibition of N-glycosylation at N370 of GluA2, a site homologous to N363 of GluA1 [13], reduced the cell surface expression ( Figure S1) [17], highlighting the importance of N-glycan at N363 for AMPAR subunit transport from the ER to the cell surface. By contrast, under the hetero-oligomeric expression conditions of GluA1 and GluA2, in which one subunit was mutated and the other was wild-type (WT), the GluA2N370 mutation significantly reduced the cell surface expression of GluA1WT [17]; GluA2WT rescued the effects of GluA1N363 mutation [20]. These results suggest a potential role for N-glycans in the regulation of the ER-exit of hetero-oligomers.…”

“…Notably, mice lacking the HNK-1 epitope exhibited learning and memory formation impairments, as well as reduced long-term potentiation in the hippocampal CA1 region [19], indicating that N-glycosylation of AMPAR is indispensable for synaptic plasticity. By establishing N-glycosylation site mutants, we found that N-glycans at N63 and N363 of GluA1 were essential for GluA1 homotetramerization and cell surface expression [20]. Similarly, the inhibition of N-glycosylation at N370 of GluA2, a site homologous to N363 of GluA1 [13], reduced the cell surface expression ( Figure S1) [17], highlighting the importance of N-glycan at N363 for AMPAR subunit transport from the ER to the cell surface.…”

Structural Insights into GluK3-kainate Receptor Desensitization and Recovery

“…The polar, charged and complex sugar chains present in Nglycans may interact with other synaptic proteins or lay in close proximity to the functional domain of iGluRs 19 thereby affecting receptor functionality such as deactivation, desensitization and recovery from desensitization 20 . N-glycans have also been shown to be important for efficient assembly and trafficking of AMPA 21,22 NMDA 23,24 and KA receptors 20 .…”

Monitoring the glycosylation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate‐type glutamate receptors using specific antibodies reveals a novel regulatory mechanism of N‐glycosylation occupancy by molecular chaperones in mice

“…In our previous study, we found that the inhibition of N‐glycosylation at the N63 or N363 site by amino acid substitution of asparagines residue caused impaired tetramer formation and suppressed cell surface transport, followed by degradation through the lysosomal pathway. (Kandel et al, ). These results suggest the importance of N‐glycans at these sites for the quality control of GluA1.…”