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Characterization of the functional role of the N‐glycans in the AMPA receptor ligand‐binding domain

The ligand‐binding domains of AMPA receptor subunits carry two conserved N‐glycosylation sites. In order to gain insight into the functional role of the corresponding N‐glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand‐binding characteristics, structural stability, cell‐surface expression, and channel properties of homomeric GluR‐D (GluR4) receptor and its soluble ligand‐binding domain (S1S2). GluR‐D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand‐binding properties were observed between the ‘wild‐type’ S1S2 and non‐glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N‐glycosylation. Purified glycosylated and non‐glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full‐length homomeric GluR‐D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild‐type GluR‐D. In outside‐out patches, GluR‐D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand‐binding domain glycans do not play any major role in receptor–ligand interactions, do not impart a stabilizing effect on the ligand‐binding domain, and are not critical for the formation and surface localization of homomeric GluR‐D AMPA receptors in HEK293 cells.