title: Biochemical and electrophysiological characterization of N-glycans on NMDA receptor subunits.
abstract: In mammals, excitatory synapses contain two major types of ionotropic glutamate receptors: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptors (NMDARs). Both receptor types are comprised of several subunits that are post-translationally modified by N-glycosylation. However, the precise N-glycans that are attached to these receptor types are largely unknown. Here, we used biochemistry to confirm that native NMDARs are extensively N-glycosylated; moreover, we found that the NMDAR GluN2B subunit differs from GluN1 subunits with respect to endoglycosidase H sensitivity. Next, we used a complete panel of lectins to determine the glycan composition of NMDARs in both cerebellar tissue and cultured cerebellar granule cells. Our experiments identified 23 lectins that pulled down both the GluN1 and GluN2B NMDAR subunits. We then performed an electrophysiological analysis using representative lectins and found that pre-incubating cerebellar granule cells with the AAL, WGA, or ConA alters the receptor's biophysical properties; this lectin-mediated effect was eliminated when the cells were deglycosylated with peptide-N-glycosidase F. Similar lectin-mediated effects were observed using HEK293 cells that express recombinant GluN1/GluN2B receptors. Finally, using mutant recombinant GluN subunits expressed in HEK293 cells, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data provide new insight into the role that N-glycosylation plays in regulating the function of NMDA receptors in the central nervous system. All animal experiments were performed in accordance with relevant institutional ethics guidelines and regulations with respect to protecting animal welfare. We examined the N-glycan composition of NMDA receptors (NMDARs) using deglycosylating enzymes, lectin-based biochemistry, and electrophysiology. Our results revealed that cerebellar NMDARs associate with 23 different lectins that have unique specificities for glycan structures. Furthermore, we found that 11 out of 12 predicted N-glycosylation sites in GluN1 and 7 out of 7 N-glycosylation sites in GluN2B are occupied by N-glycans. These data shed light on the glycan composition of NMDARs, revealing potential targets for the development of novel therapeutic approaches.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Prague 4, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Kaniakova
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Prague 4, Czech Republic.
authors.forename: Katarina
authors.initials: K
authors.lastname: Lichnerova
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Prague 4, Czech Republic.|Department of Physiology, Faculty of Science, Charles University in Prague, Albertov 6, Czech Republic.
authors.forename: Kristyna
authors.initials: K
authors.lastname: Skrenkova
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Prague 4, Czech Republic.
authors.forename: Ladislav
authors.initials: L
authors.lastname: Vyklicky
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Prague 4, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Horak
mesh_terms: D000818:Animals; D055724:Electrophysiological Phenomena / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013569:Synapses / Q000378:metabolism*
chemical_list: C121001:NR2B NMDA receptor; D011134:Polysaccharides; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid
keywords: biochemistry; cerebellar granule cells; glutamate receptor; ion channel; patch-clamp