Functional assessment of triheteromeric NMDA receptors containing a human variant associated with epilepsy.

Katie F M Marwick,Paul A Skehel,Giles E Hardingham,Kasper B Hansen,David J A Wyllie

doi:10.1113/jp277292

Katie F M Marwick, Paul A Skehel + Show 3 more

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https://doi.org/10.1113/jp277292

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Abstract

Key points NMDA receptors are neurotransmitter‐gated ion channels that are critically involved in brain cell communicationVariations in genes encoding NMDA receptor subunits have been found in a range of neurodevelopmental disorders.We investigated a de novo genetic variant found in patients with epileptic encephalopathy that changes a residue located in the ion channel pore of the GluN2A NMDA receptor subunit.We found that this variant (GluN2AN615K) impairs physiologically important receptor properties: it markedly reduces Mg2+ blockade and channel conductance, even for receptors in which one GluN2AN615K is co‐assembled with one wild‐type GluN2A subunit.Our findings are consistent with the GluN2AN615K mutation being the primary cause of the severe neurodevelopmental disorder in carriers. NMDA receptors are ionotropic calcium‐permeable glutamate receptors with a voltage‐dependence mediated by blockade by Mg2+. Their activation is important in signal transduction, as well as synapse formation and maintenance. Two unrelated individuals with epileptic encephalopathy carry a de novo variant in the gene encoding the GluN2A NMDA receptor subunit: a N615K missense variant in the M2 pore helix (GRIN2A C1845A). We hypothesized that this variant underlies the neurodevelopmental disorders in carriers and explored its functional consequences by electrophysiological analysis in heterologous systems. We focused on GluN2AN615K co‐expressed with wild‐type GluN2 subunits in physiologically relevant triheteromeric NMDA receptors containing two GluN1 and two distinct GluN2 subunits, whereas previous studies have investigated the impact of the variant in diheteromeric NMDA receptors with two GluN1 and two identical GluN2 subunits. We found that GluN2AN615K‐containing triheteromers showed markedly reduced Mg2+ blockade, with a value intermediate between GluN2AN615K diheteromers and wild‐type NMDA receptors. Single‐channel conductance was reduced by four‐fold in GluN2AN615K diheteromers, again with an intermediate value in GluN2AN615K‐containing triheteromers. Glutamate deactivation rates were unaffected. Furthermore, we expressed GluN2AN615K in cultured primary mouse cortical neurons, observing a decrease in Mg2+ blockade and reduction in current density, confirming that the variant continues to have significant functional impact in neuronal systems. Our results demonstrate that the GluN2AN615K variant has substantial effects on NMDA receptor properties fundamental to the roles of the receptor in synaptic plasticity, even when expressed alongside wild‐type subunits. This work strengthens the evidence indicating that the GluN2AN615K variant underlies the disabling neurodevelopmental phenotype in carriers.

Highlights

The NMDA receptor is an ionotropic voltage-dependent glutamate receptor with important roles in synaptic signal transduction and plasticity
We investigated the impact of the GluN2AN615K variant when expressed as part of triheteromeric NMDA receptors containing wild-type GluN2 subunits (Fig. 1)
We investigated the functional consequences of GluN2AN615K, a heterozygous missense variant found to have arisen de novo in two unrelated people with early onset epileptic encephalopathy

Summary

Introduction

The NMDA receptor is an ionotropic voltage-dependent glutamate receptor with important roles in synaptic signal transduction and plasticity. It is improbable that all variants are disease-causing: some are inherited from phenotypically normal parents and some are in regions of the protein that probably exhibit variation without deleterious functional impact (less highly conserved, or more variation present in healthy controls) Of those variants where functional consequences have been assessed, a range of effects have been found, some predominantly ‘gain-of-function’, some predominantly ‘loss-of-function’ (Swanger et al 2016) and some with no effect (Marwick et al 2017). No previous work has investigated the impact of this variant when expressed in triheteromers with one wild-type and one mutated GluN2 subunit This insight would be clinically relevant, both because the variant is present heterozygously and because the majority of NMDA receptors in key regions of the adolescent and adult brain are probably GluN1/GluN2A/GluN2B triheteromers. The mutations were verified by Sanger sequencing through the mutated region

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