Conservation of human NMDA receptor subunits and disease variants in zebrafish

NMDA receptors (NMDARs) are widely expressed, glutamate-gated ion channels that play key roles in brain development and function. Variants have been identified in the GRIN genes encoding NMDAR subunits that are linked to neurodevelopmental disorders, among other manifestations. Zebrafish are a powerful model to study brain development and function given their rapid development and ease of genetic manipulation. As a result of an ancient genome duplication, zebrafish possess two paralogues for most human NMDAR subunits. To evaluate the degree of conservation between human NMDAR subunits and their respective zebrafish paralogues, we carried out detailed in silico analyses, with an emphasis on key functional elements. To further assess the suitability of zebrafish for modeling NMDAR-associated neurodevelopmental disorders, we analyzed the conservation of positions with identified missense variants.

We find that the human NMDAR subunits are generally well conserved across zebrafish paralogs. Moreover, variants classified as pathogenic and putatively pathogenic are highly conserved, reflecting the importance of key protein regions to neurotypical receptor function. Positions with putatively benign and benign variants are less conserved. Across NMDAR domains, the transmembrane domain is most highly conserved, followed by the ligand-binding domain, which maintains conservation of amino acids that participate in the binding of ligands. The N-terminal domain is less well conserved but aligned homology models show high degrees of structural similarity. The C-terminal domain is the most poorly conserved region across zebrafish paralogs, but certain key regions that undergo phosphorylation, palmitoylation, and ubiquitylation as well as protein-binding motifs are better conserved.

Our findings highlight a strong conservation of human NMDAR subunits in zebrafish, with some exceptions. The ligand-binding domain, the transmembrane domain forming the ion channel and the short polypeptide linkers that connect them are highly conserved. The N- and C-terminal domains are less conserved but functional motifs in general, except for the Zn²⁺ binding site in GluN2A paralogues, are more highly conserved relative to the entire domain. Overall, our findings support the utility of zebrafish as a model for studying neurodevelopment and disease mechanisms and provide a template for rigorously considering the relationship between human and zebrafish paralogues.