AMPA-type glutamate receptors (AMPAR) are hetero or homotetrameric complexes composed of various combinations of four subunits (GluA1-4) and mediate most of the fast excitatory synaptic transmission in the nervous system. Postsynaptic cell surface expression level of AMPAR determines synaptic plasticity, which contributes to a basis of memory formation and learning. The abundance of AMPAR at postsynaptic region is regulated by the combination of several events including the trafficking from the intracellular AMPAR to cell surface and lateral movements between extrasynaptic and synaptic region. Study on these phenomena thus yields comprehensive understandings of a neuronal activity at the molecular level.
So far, we found that human natural killer-1 (HNK-1) carbohydrate, specifically expressed on N-linked glycans of GluA2, regulated cell surface expression level of AMPAR and spine maturation process. We considered that other N-glycans expressed on AMPAR also play important roles in regulating functions of AMPAR such as the trafficking and lateral movement. So we first investigated cell surface expression levels of a series of GluA1 and GluA2 mutants, of which an Asn residue in the consensus sequence (N-X-S/T) was mutated to Ser, with a cell surface biotinylation assay. As a result, cell surface expression levels of GluA1N63S, GluA1N363S, and GluA2N370S were decreased compared with those of wild-type, and these mutants were mainly distributed in ER, suggesting that these N-glycans have important roles in the trafficking of AMPAR. Next, we applied advanced single-molecule fluorescent imaging to directly observe the movement of AMPAR on cell surface membrane and tried to investigate the role of N-glycans. For the imaging, we constructed ACP-tagged and Halo-tagged GluA1 and GluA2 and revealed with patch-clamp method that those tags did not influence on the channel activity. We will present the recent observation by using this imaging system.