The structural changes of the ligand-binding domains within the GluN1 and GluN2A subunits regulate the surface delivery and function of NMDA receptors in mammalian cells
The seminar will take place on 7. 9. 2023 from 2 p.m. in the Turquoise Auditorium of the IEM CAS.
N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission and synaptic plasticity. An important measure of the functionality of NMDARs is their number on the neuronal surface, but this process including quality control in the endoplasmic reticulum (ER) is not fully elucidated. In our studies, we focused on investigating the molecular mechanisms in the early trafficking of the most abundant type of the NMDAR in the adult cortex, GluN1/GluN2A, by using substitutions of amino acids interacting in ligand binding domains with endogenous ligands, i.e., glycine in the GluN1 subunit and L-glutamate in the GluN2A subunit.
Our microscopical and electrophysiological experiments showed that alanine substitutions in both subunits substantially increased the EC50 values for glycine or L-glutamate, and that these changes correlated with lower surface expression of GluN1/GluN2A receptors. Using time-resolved release from the ER, we showed that alanine substitutions in LBDs altered the release of the GluN1/GluN2A receptors from the ER. We further showed that co-expression of two alanine substitutions in the LBDs of the GluN1 and GluN2A subunits additively reduces the surface expression of the GluN1/GluN2A receptors compared to receptors carrying only one mutated subunit. Finally, we characterised the mutation cycles for GluN1-S688 and GluN1-D732 residues, including the pathogenic S688P, S688Y and D732E variants. These experiments showed that different amino acid substitutions at the same positions of the GluN1 subunit substantially altered the surface numbers of NMDARs, which in most cases correlated with their sensitivity to glycine. Our study helps to understand the underlying mechanisms regulating the early transport of NMDARs while characterising the impact of a set of pathogenic variants on the regulation of their surface expression.
This work was supported by the Czech Science Foundation (GAČR: 20-12420S) and the Charles University Grant Agency (GAUK: 306221).
We look forward to your participation.