Functional Evaluation of a De Novo GRIN2A Mutation Identified in a Patient with Profound Global Developmental Delay and Refractory Epilepsy

Abstract

The N-methyl-d-aspartate receptor (NMDAR), a ligand-gated ionotropic glutamate receptor, plays important roles in normal brain development and a wide range of neurologic disorders, including epilepsy. Here, we evaluate for the first time the functional properties of a de novo missense mutation (p.M817V) in the pre-M4 linker in a child with profound global developmental delay and refractory epilepsy. Electrophysiologic recordings revealed that the mutant GluN2A(M817V)-containing receptors showed enhanced agonist potency, reduced sensitivity to endogenous negative inhibitors (Mg, proton, and zinc), prolonged synaptic-like response time course, increased single-channel mean open time, and increased channel open probability. These results suggest that the gain-of-function M817V mutation causes overactivation of NMDAR and drives neuronal hyperexcitability, which may contribute to the patient's observed epileptic phenotype. Molecular modeling of the closed channel conformation reveals that this mutation weakens the interaction between GluN2 transmembrane helix M4 and two GluN1 transmembrane helices, and increases atomic fluctuation or movement of the pre-M1 region of GluN1 subunit, suggesting a mechanism by which channel function is enhanced. The functional changes of this mutation on agonist potency occur when the mutation is introduced into all other GluN2 subunits, suggesting a conserved role of this residue in control of NMDAR function through interactions of membrane spanning GluN2 and GluN1 helices. A number of NMDAR-targeted drugs including U.S. Food and Drug Association-approved NMDAR channel blockers were evaluated for their ability to inhibit receptors containing GluN2A(M817V) as a first step to exploring the potential for rescue pharmacology and personalized medicine.

Structural modeling for the open state of an NMDA receptor

“…Electrophysiological studies have shown significant impairment in channel gating properties by mutations in these positions (Chen et al, 2017; Ogden et al, 2017). Moreover, the external half of the M4 helix, partly via intrasubunit interactions with the membrane-parallel pre-M1 helix, contributes significantly to channel activation (Amin et al, 2017).…”

“…(Zhou and Cross, 2013). The lack of an open state structure has greatly hampered mechanistic understanding of iGluRs (Amin et al, 2017; Chen et al, 2017; Ogden et al, 2017; Zhou and Wollmuth, 2017). …”

A novel missense mutation in GRIN2A causes a nonepileptic neurodevelopmental disorder

“…The whole‐cell voltage clamp current recordings were performed on transiently transfected human embryonic kidney cells 293 (HEK293) (ATCC CRL‐1573, Manassas, Virginia) with plasmid cDNAs encoding WT human GluN1/GluN2A or GluN1/GluN2A‐A643D with a solution containing (in mM) 150 NaCl, 3 KCl, 10 HEPES (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid), 0.01 EDTA, 1.0 CaCl 2 (calcium chloride), and 11 D‐mannitol, with the pH adjusted to 7.4 by the addition of NaOH (sodium hydroxide) (23 °C) . The recording electrodes were prepared a vertical puller (Narishige P‐10, Tokyo, Japan) using thin‐walled filamented borosilicate glass (TW150F‐4; World Precision Instruments, Sarasota, Florida) and filled with the internal solution (in mM: 110 D‐gluconic acid, 110 CsOH (cesium hydroxide), 30 CsCl (cesium chloride), 5 HEPES, 4 NaCl, 0.5 CaCl 2 , 2 MgCl 2 , (magnesium chloride) 5 BAPTA (1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid), 2 Na 2 ATP (adenosine 5′‐triphosphate disodium), 0.3 NaGTP (guanosine 5′‐triphosphate sodium); pH 7.35 with 300‐310 mOsmol/kg of the osmolality).…”

“…Finally, Carvill and colleagues identified GRIN2A mutations in 9% (4/44) of patients with epilepsy‐aphasia spectrum. Alterations in GRIN2A have also been reported in patients with early‐onset epileptic encephalopathy and severe developmental delay . To date, whole‐exome and genome sequencing have identified a substantial number of point mutations and deletions (>100) scattered across all domains of the GRIN2A gene .…”

A conserved glycine harboring disease-associated mutations is required for slow deactivation and high Ca2+ permeability in NMDA receptors

“…In the NMDAR M4 segments, there are numerous missense mutations associated with clinical pathologies (Figures 1b & 1c; Supplementary Table 1). These mutations are primarily located in the extreme extracellular third of the M4 segments (Figure 1c), which participates strongly in ion channel gating 15,16 , implicating gating deficits as a potential cause for these disease phenotypes.…”