Abstracts

Rationale: N-methyl-D-aspartate (NMDA) receptors play important roles in various pathological conditions, including epilepsy. A novel de novo GRIN2A missense mutation (p.L812M) was identified in a child with refractory seizures and epileptic encephalopathy. The functional properties of the GluN2A receptor bearing this mutation (GluN2A-L812M) are evaluated compared with those of the wild type (WT) human GluN2A receptors. Methods: Site-directed mutagenesis was performed using the QuickChange protocol to generate human GluN2A-L812M construct. Preparation of cRNA and two-electrode voltage-clamp recordings from Xenopus laevis oocytes were performed as previously described (see Traynelis et al., J Neurosci. 1998; 18(16):6163-75 for methods). The recording solution contained (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl2, 0.01 EDTA (pH 7.4). The agonist-induced accessibility changes for the sulfhydryl-modifying agent MTSEA were evaluated and the channel open probability (P_OPEN) was calculated (see Yuan et al., J Biol Chem. 2005;280(33):29708-16; Yuan et al., J Neurosci. 2009;29:12045-58 for methods)Results: The pharmacological properties of this mutant receptor were compared with WT receptors by voltage clamp recordings in vitro. GluN2A-L812M-containing receptors showed an 8-fold increase in potency for glutamate (EC₅₀ 0.43 M vs. 3.4 M of WT) as well as glycine (EC₅₀ 0.13 M vs. 1.0 M of WT). This result suggested that the patient s NMDARs could be activated by lower concentrations of these co-agonists than typically occur at the synapse. Voltage-dependent Mg²⁺ block is another important feature of NMDAR activity. Analysis of the current-voltage relationship indicated a diminished Mg²⁺ block resulting in more current through the mutant L812M receptors (60% vs. 33% of WT at -30 mV; 50% vs. 21% at -45 mV). The mutant L812M receptors reduced proton inhibition (IC₅₀ pH 6.5 vs. pH 6.9 of WT) and also decreased both the potency (IC₅₀ 1243 nM vs. 65 nM of WT) and the degree of zinc inhibition (maximal inhibition 6% vs. 44% of WT). These data suggested that the mutant L812M receptors are less sensitive to the negative modulators. The channel open probability calculated for the mutant receptors was 0.38, which is 2-fold higher than 0.19 observed for the wild type, and indicates that the mutant channels contribute to profound NMDA receptor hyperactivity by being open more often.Conclusions: Our results indicate that, based upon electrophysiological testing, mutant NMDARs have enhanced sensitivity to agonists, decreased effects of endogenous negative modulators, and increased channel open probability. These gain-of-function changes may result in refractory seizures and profound cognitive impairment. These results suggest that future evaluations of children with refractory seizures and early-onset epileptic encephalopathies might include evaluation of NMDAR subunit genes. We are optimistic that further studies of the genetics and pathophysiology of NMDA receptor channelopathies will lead to improved therapies for the medically intractable epilepsies.