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

Abstract

Key points NMDA receptors are neurotransmitter‐gated ion channels that are critically involved in brain cell communication Variations 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 (GluN2A N615K ) impairs physiologically important receptor properties: it markedly reduces Mg 2+ blockade and channel conductance, even for receptors in which one GluN2A N615K is co‐assembled with one wild‐type GluN2A subunit. Our findings are consistent with the GluN2A N615K mutation being the primary cause of the severe neurodevelopmental disorder in carriers. Abstract NMDA receptors are ionotropic calcium‐permeable glutamate receptors with a voltage‐dependence mediated by blockade by Mg 2+ . 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 GluN2A N615K 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 GluN2A N615K ‐containing triheteromers showed markedly reduced Mg 2+ blockade, with a value intermediate between GluN2A N615K diheteromers and wild‐type NMDA receptors. Single‐channel conductance was reduced by four‐fold in GluN2A N615K diheteromers, again with an intermediate value in GluN2A N615K ‐containing triheteromers. Glutamate deactivation rates were unaffected. Furthermore, we expressed GluN2A N615K in cultured primary mouse cortical neurons, observing a decrease in Mg 2+ blockade and reduction in current density, confirming that the variant continues to have significant functional impact in neuronal systems. Our results ...

De novoGRINvariants in NMDA receptor M2 channel pore‐forming loop are associated with neurological diseases

“…A second important conclusion from this study is that the M2 region can influence a host of receptor properties in addition to Mg 2+ block and Ca 2+ permeability, including agonist potency, proton sensitivity, open probability, and response time course, consistent with the recent studies (Fedele et al, ; Marwick, Hansen, Skehel, Hardingham, & Wyllie, ; Marwick, Skehel, Hardingham, & Wyllie, ; Mullier et al, ). For some variants, multiple functional effects have seemingly opposite actions when viewed simplistically as either enhancing or decreasing NMDAR function.…”

The human NMDA receptor GluN2AN615K variant influences channel blocker potency

“…All cDNAs were in pCI‐neo. Site‐directed mutagenesis to generate GluN2A N615K was performed as described previously using a mutagenizing polymerase chain reaction, recircularization and transformation. The mutation was verified using Sanger sequencing through the mutated region.…”

“…We examined the previously uninvestigated blocker ketamine. In addition, we replicated our previous finding of a reduction in single‐channel conductance in a different system using a different method of measurement. Our findings show that blocking GluN2A N615K –containing NMDA receptors using memantine or amantadine remains possible in the presence of Mg 2+ , but that dextromethorphan is a more promising therapeutic candidate due to its increased inhibition in the presence of the variant.…”

“…We found that GluN2A N615K –containing NMDA receptors showed a 4‐fold reduction in single–channel conductance. The conductance value obtained here using technically “gold standard” outside‐out patches is in good agreement with that obtained using cell–attached patches containing GluN2A N615K diheteromeric receptors expressed in human embryonic kidney (HEK293T) cells . The reduction in cation conductance seen likely reflects the introduction of a positively charged residue at a constriction of the pore .…”

“…Previous work has shown that the GluN2A N615K variant has profound effect on NMDA receptor properties: it reduces block by Mg 2+ and influences block by other channel blockers, it reduces calcium permeability and it reduces single–channel conductance . Importantly, the variant influences receptor properties even when only one copy is present in a receptor . Some of these effects could be viewed as “gain of function”, some “loss of function”.…”