Abstracts

Rationale: NMDA receptors mediate a slow component of excitatory synaptic transmission in the brain. Overactivation of these receptors can be harmful due to swelling, which is secondary to influx of Na+, and excessive accumulation of intracellular Ca2+. Recently, mutations in the genes encoding NMDA receptor subunits have been identified in a number of cases of developmental delay and epilepsy, with mutations in the GRIN2A gene encoding the GluN2A subunit being most common. Here we describe functional changes in GRIN2A pre-M1 mutation (GluN2A-P552R), which was identified in a patient with developmental delay and epilepsy (de Ligt et al., 2012).Methods: Site-directed mutagenesis, preparation of cRNA, and two-electrode voltage-clamp recordings from Xenopus oocytes were performed as previously described (Traynelis et al., 1998). The response time course was evaluated by whole cell voltage-clamp current recordings on transfected HEK cells by a rapid solution exchange system (Yuan et al., 2005; 2009).Results: We evaluated the functional effects of this mutation on recombinant GluN1/GluN2A receptors, and found that the potency of both glutamate (EC50 0.37 μM vs. 3.3 μM of WT; n=9-13) and glycine (EC50 0.06 μM vs. 1.2 μM of WT; n=10) increased ~10-fold without a change in open probability (n=12-17). The response time course to rapid application of glutamate was dramatically slowed, with the 10-90% rise time increased 50-fold from 10 to 576 ms, and the weighted time constant describing the deactivation time course following rapid removal of glutamate increased from 45 to 794 ms (n=5-6). As a result of these changes, charge transfer in response to brief synaptic-like exposure to glutamate increased 25-fold, raising the possibility that this mutation leads to neurotoxicity. To assess this possibility, we transfected rat cortical neuronal cultures with GFP+vector, GFP+ WT GluN2A, or GFP+GluN2A-P552R at 21-25 days in vitro. Our preliminary results indicate that neuronal morphology was unchanged between GFP+vector and GFP+GluN2A-transfected neurons; however transfection of GFP+GluN2A-P552R produced extensive swelling within the dendrites. Quantification of co-transfected luciferase suggested mutant GluN2A-P552R increased neuronal death. The influence of this mutation on sensitivity to FDA-approved NMDAR antagonists, including memantine, dextromethorphan, and amantadine was also evaluated. The results raise the possibility an NMDAR antagonist might be useful to attenuate NMDAR overactivation, which might decrease seizure burden in these patients.Conclusions: These data suggest that the GluN2A-P552R mutant NMDAR has enhanced sensitivity to agonists and a prolonged synaptic response time course, which may contribute to the neuropathology observed in this patient. NMDAR antagonists should reduce NMDAR overactivation and may constitute useful therapies for associated intractable seizures.