Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) play important roles in a wide range of neurological disorders, including epilepsy. A de novo GRIN2A missense mutation (p.M817V) has been reported in a child with severe global developmental delay and intractable epilepsy (Venkateswaran et al., 2014). To evaluate the contribution of this mutation to clinical phenotype, we compared the functional properties of GluN2A-M817V-containing NMDARs to wild type (WT) GluN2A receptors.Methods: The human GluN2A-M817V cDNA was generated by using the QuikChange protocol for site-directed mutagenesis. Preparation of cRNA and two-electrode voltage-clamp recordings from Xenopus laevis oocytes were performed as previously described (see Traynelis et al., 1998 for Methods). The synaptic response time course was estimated by a rapid solution exchange system, which allowed brief (1-4 ms) application of maximally effective glutamate. We recorded current responses using whole cell voltage-clamp from transfected HEK cells (see Yuan et al., 2005; 2009 for Methods).Results: The pharmacological properties of this mutant receptor were compared with WT receptors by voltage clamp recordings from Xenopus laevis oocytes. GluN2A-M817V-containing NMDARs showed an 11-fold increase in potency for glutamate (EC50 0.30 μM vs. 3.6 μM of WT) and a 9-fold increase in potency for glycine (EC50 0.10 μM vs. 0.90 μM of WT). This result suggested that the patient’s NMDARs could be activated by lower concentrations of these co-agonists than observed for wild type receptors. The response time course (tau deactivation rate) to brief synaptic-like application of glutamate for the mutant was 632 ms, which is 15-fold longer than 43 ms observed for wild type receptors, and suggests that the mutant channels contribute to NMDAR hyperactivity by prolonging the synaptic response. The influence of this mutation on sensitivity to FDA-approved NMDAR antagonists (e.g. memantine, dextromethorphan, and amantadine) was also evaluated. The results raise the possibility that an NMDAR antagonist might attenuate NMDAR overactivation, which might decrease seizure frequency or severity in these patients, emphasizing the potential opportunity for personalized medicine.Conclusions: The data indicate that NMDARs containing the missense mutation GluN2A-M817V show enhanced sensitivity to agonists and a prolonged response time course to brief synaptic-like application of glutamate. This gain-of-function may contribute to seizures and cognitive problem. We suggest that further studies of the genetics and pathophysiology of NMDAR channelopathies will lead to improved therapies for the epileptic syndrome.