Abstract
Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and d-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.
Highlights
Numerous pathogenic mutations have been identified in various subunits of N-methyl-Daspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown
With respect to the interaction between d-serine and the ligand-binding domain (LBD) in wildtype GluN1, our model suggests that the d-serine molecule likely forms hydrogen bonds between its functional carboxyl group and the guanidinium moiety in the R523 residue and the backbone amides in S688 and T518 (Fig. 1b)
We focused our study on NMDARs in the mammalian central nervous system (CNS), as these receptors play an important role in a wide range of physiological processes such as learning and memory[56,57], as well as neuropathological processes such as n eurodegeneration[54]
Summary
Numerous pathogenic mutations have been identified in various subunits of N-methyl-Daspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Most studies suggest that the intracellular CTD of GluN subunits plays a critical role in regulating the surface expression of NMDARs, including receptors that contain GluN2A and/or GluN2B s ubunits[5]. We used in silico modelling as well as electrophysiology, microscopy, and biochemistry in HEK293 cells and primary rat hippocampal neurons to examine the effect of the pathogenic S688Y mutation in the LBD of the GluN1 subunit on the trafficking and function of NMDARs. We found that the GluN1-S688Y subunit alters the functional properties of GluN1/ GluN2A, GluN1/GluN2B, and GluN1/GluN3A receptors when expressed in both HEK293 cells and hippocampal neurons. We found that the S688Y mutation reduces both NMDA-induced C a2+ influx and excitotoxicity in hippocampal neurons Taken together, these findings reveal new insights in the role that this pathogenic mutation plays in regulating various NMDAR properties
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