Two de novo GluN2B mutations affect multiple NMDAR-functions and instigate severe pediatric encephalopathy

The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at excitatory synapses. Mutations affecting receptor function often lead to devastating neurodevelopmental disorders. We have identified two toddlers with different heterozygous missense mutations of the same, and highly conserved, glycine residue located in the ligand-binding-domain of GRIN2B: G689C and G689S. Structure simulations suggest severely impaired glutamate binding, which we confirm by functional analysis. Both variants show three orders of magnitude reductions in glutamate EC₅₀, with G689S exhibiting the largest reductions observed for GRIN2B (~2000-fold). Moreover, variants multimerize with GluN2Bwt and GluN2Awt-subunits, thus engendering a strong dominant-negative effect on mixed channels. In neurons, overexpression of the variants instigates suppression of synaptic GluNRs, an effect which was reversed by the positive allosteric modulator, pregnenolone. Lastly, while exploring spermine potentiation as a potential treatment, we discovered that the variants fail to respond due to G689’s novel role in proton-sensing. Together, we describe two unique variants with extreme effects on channel function and purpose a potential treatment.