Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) play important roles in various pathological conditions, including epilepsy. A de novo GRIN2A missense mutation (p.K669N) has been reported in a child with CSWSS, Continuous Spike and Waves during Slow-wave Sleep Syndrome (Lesca et al., 2013). The functional properties of the GluN2A receptor bearing this mutation (GluN2A-K669N) are evaluated compared with those of the wild type (WT) human GluN2A receptors. Methods: Site-directed mutagenesis was performed using the QuikChange protocol to generate human GluN2A-K669N construct. 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 response time course was evaluated by whole cell voltage-clamp current recordings on transfected HEK cells by a rapid solution exchange system (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 in vitro. GluN2A-K669N-containing NMDARs showed an 5-fold increase in potency for glutamate (EC₅₀ 0.70 μM vs. 3.4 μM of WT) and a 10-fold increase in potency for glycine (EC₅₀ 0.10 μM vs. 1.0 μM of WT). This result suggested that the patient's NMDARs could be activated by lower concentrations of these co-agonists than typically occur for wild type receptors. The synapse response time course (tau deactivation rate) for the mutant was 235 ms, which is 4-fold longer than 58 ms observed for wild type, and suggests that the mutant channels contribute to NMDAR hyperactivity by prolonging synaptic response. 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 alternate NMDAR overactivation, which might decrease seizure frequency or severity in these patients, emphasizing the potential realization of personalized medicine. Conclusions: Our results indicate that, based upon electrophysiological testing, the GluN2A-K669N mutant NMDAR has enhanced sensitivity to agonists. Our data predict a prolonged synapse response time course. These gain-of-function changes may contribute to seizures and cognitive impairment. We are optimistic that further studies of the genetics and pathophysiology of NMDA receptor channelopathies will lead to improved therapies for the epileptic syndrome.