The human NMDA receptor GluN2AN615K variant influences channel blocker potency

Abstract N‐methyl‐D‐aspartate (NMDA) receptors are glutamate receptors with key roles in synaptic plasticity, due in part to their Mg2+ mediated voltage‐dependence. A large number of genetic variants affecting NMDA receptor subunits have been found in people with a range of neurodevelopmental disorders, including GluN2AN615K (GRIN2A C1845A) in two unrelated individuals with severe epileptic encephalopathy. This missense variant substitutes a lysine in place of an asparagine known to be important for blockade by Mg2+ and other small molecule channel blockers. We therefore measured the impact of GluN2AN615K on a range of NMDA receptor channel blockers using two‐electrode voltage clamp recordings made in Xenopus oocytes. We found that GluN2AN615K resulted in block by Mg2+ 1 mmol/L being greatly reduced (89% vs 8%), block by memantine 10 μmol/L (76% vs 27%) and amantadine 100 μmol/L (45% vs 17%) being substantially reduced, block by ketamine 10 μmol/L being modestly reduced (79% vs 73%) and block by dextromethorphan 10 μmol/L being enhanced (45% vs 55%). Coapplying Mg2+ with memantine or amantadine did not reduce the GluN2AN615K block seen with either small molecule. In addition, we measured single–channel conductance of GluN2AN615K–containing NMDA receptors in outside‐out patches pulled from Xenopus oocytes, finding a 4‐fold reduction in conductance (58 vs 15 pS). In conclusion, the GluN2AN615K variant is associated with substantial changes to important physiological and pharmacological properties of the NMDA receptor. Our findings are consistent with GluN2AN615K having a disease–causing role, and inform potential therapeutic strategies.


| INTRODUC TI ON
N-methyl-D-aspartate receptors are calcium-permeable ligandgated glutamate receptors which unusually are blocked at rest by Mg 2+ ions and thus also require postsynaptic depolarization in order to allow current flow. 1,2 This dual requirement for activation affords them crucial roles in synapse formation, plasticity and maintenance. 3 Consistent with these important roles, NMDA receptor dysfunction has been linked to a range of neurodevelopmental and neurodegenerative disorders 3  NMDA receptors are heterotetramers containing two obligatory GluN1 subunits and two others, of which GluN2A and GluN2B are the commonest in the postnatal mammalian forebrain. 5 Where two types of subunits are present the receptor is termed a diheteromer, where three types are present, a triheteromer. Over the last decade a large and increasing number of variants in the genes encoding NMDA receptor subunits have been identified in people with neurodevelopmental disorders including intellectual disability, schizophrenia, autism and epilepsy. 6 Variants in GRIN2A are most likely to be associated with epilepsy aphasia syndromes, with the location and nature of the variant (loss of function vs missense) influencing the severity of the phenotype seen. 7 The missense variant GluN2A N615K is associated with a severe phenotype of early onset epileptic encephalopathy in two unrelated individuals. 8,9 It substitutes a lysine (positively charged) for an asparagine (neutral) in the M2 region of the NMDA receptor pore, in one of the narrowest regions of the pore. 10 The residue affected is the most important determinant of Mg 2+ block in GluN2A subunits: the "N + 1" site (an asparagine that neighbors the QRN site asparagine in GluN2A). 11 Previous work has shown that the GluN2A N615K variant has profound effect on NMDA receptor properties: it reduces block by Mg 2+4, 9,12 and influences block by other channel blockers, 4 it reduces calcium permeability 9 and it reduces single-channel conductance. 12 Importantly, the variant influences receptor properties even when only one copy is present in a receptor. 12 Some of these effects could be viewed as "gain of function", some "loss of function". Seeking to reverse the "gain of function" component could be aided by the use of channel blockers, as has been trialed successfully by the use of memantine in a child carrying a different GRIN2A variant. 4 To do this, detailed knowledge of the effect of channel blockers on receptors containing the GluN2A N615K variant in physiological contexts is required.
In this study we therefore sought to replicate and extend previous work demonstrating a reduced potency of memantine and amantadine, 4 by investigating the degree of inhibition by these blockers in the presence and absence of physiological concentrations of Mg 2+ . We examined the previously uninvestigated blocker ketamine. In addition, we replicated our previous finding of a reduction in single-channel conductance 12 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.

| cRNA synthesis and expression in oocytes
The cDNA for wild type human NMDA subunit GluN1-1a (hereafter GluN1) and GluN2A (GenBank accession codes: NP_015566, NP_000824) 13 were gifts from Dr Hongjie Yuan (University of Emory). All cDNAs were in pCI-neo. Site-directed mutagenesis to generate GluN2A N615K was performed as described previously 12 using a mutagenizing polymerase chain reaction, recircularization and transformation. The mutation was verified using Sanger sequencing through the mutated region.
cRNA synthesis and expression was performed as described previously. 14 cRNA for wild type and mutant subunits was synthesized from

| Two-electrode voltage-clamp recordings
Two-electrode voltage-clamp recordings were performed as described previously. 14 Recordings were made at room temperature

| Single-channel voltage-clamp recordings
Single-channel voltage-clamp recordings were made as described previously. 14 Recordings were made at room temperature (18-21°C) Patches were voltage-clamped at -60 or -100 mV.

Bar graphs depict individual cells (circles), means (columns) and
standard error of the mean (error bars). R (v 3.1.2) (R Core Team, 2014) was used to perform statistical tests. Comparisons between multiple means were performed using ANOVA, with post hoc tests performed if the F test was significant for a main effect. Comparisons between two means were performed using independent, two-tailed, Welch t-tests (which do not assume equal variance between groups) unless otherwise stated. Correction for multiple comparisons were made using the Bonferroni method. The significance level used was P < 0.05. In figures, * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001.

| Compliance with design and statistical analysis requirements
Oocytes were randomly allocated for injection with either WT or mutant cDNA. The experimenter was not blinded to construct as this was thought unlikely to influence the electrophysiological recordings. All n refer to number of oocytes, not number of traces. Although groups were designed to have equal n values, application of exclusion rules following recording (see above) meant that groups ended up unequal in size. The t-tests used for comparisons adjust for resulting unequal variance. In the case of single-channel recordings the n numbers reported are less than 5 but data obtained are highly consistent.

| Drugs, reagents and other materials
Memantine, amantadine, ketamine, and dextromethorphan were purchased from Tocris Bioscience (Bristol, UK). The remaining substances were purchased from Sigma-Aldrich (St Louis, MO).

| GluN2A N615K alters the inhibition of a range of NMDA receptor channel blockers, including Mg 2+
To assess the impact of the GluN2A N615K mutation on inhibition by Mg 2+ and small molecule channel blockers, we made two-electrode voltage-clamp recordings from oocytes expressing GluN2A WT or GluN2A N615K diheteromeric NMDA receptors exposed to glutamate (30 μmol/L) and glycine (30 μmol/L) while voltage-clamped at -60 mV.
We found that block by an approximately physiological concentra-

| GluN2A N615K reduces single-channel conductance
In view of the profound influence of the GluN2A N615K mutation on channel blockade we next assessed the variant's impact on ion permeation. In outside-out patches pulled from oocytes expressing GluN2A WT or GluN2A N615K -containing NMDA receptors we identified a 4-fold reduction in single-channel conductance (Figure 3).

| GluN2A N615K influences degree of blockade by NMDA receptor channel blockers
We found that GluN2A N615K reduced the inhibition caused by the small molecule channel blockers memantine, amantadine and ketamine, while increasing the inhibition caused by dextromethorphan.

| GluN2A N615K reduces single-channel conductance
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. 12 The reduction in cation conductance seen likely reflects the introduction of a positively charged residue at a constriction of the pore. 10 This region has already been shown to determine conductance by mutation studies in GluN1 and GluN2B subunits. [19][20][21] The reduction in conductance is likely to disrupt the receptor's ionotropic signaling function and thus potentially impair both neurotransmission and plasticity. However, a reduction in conductance is unlikely to be amenable to therapeutic modification by existing small molecules.
In conclusion, this study found that the human disease-associated variant GluN2A N615K has marked effects on inhibition by channel blockers and single-channel conductance. The channel blocker dextromethorphan is a potential candidate for possible therapeutic use.

ACK N OWLED G EM ENTS
We thank Dr Matt Livesey for technical assistance with generation and analysis of single-channel recordings.