Abstract
N-methyl-D-aspartate receptors (NMDARs), a subtype of glutamate-gated ion channels, play a central role in epileptogenesis. Recent studies have identified an increasing number of GRIN2A (a gene encoding the NMDAR GluN2A subunit) mutations in patients with epilepsy. Phenotypes of GRIN2A mutations include epilepsy-aphasia disorders and other epileptic encephalopathies, which pose challenges in clinical treatment. Here we identified a heterozygous GRIN2A mutation (c.1341T>A, p.N447K) from a boy with Rolandic epilepsy by whole-exome sequencing. The patient became seizure-free with a combination of valproate and lamotrigine. Functional investigation was carried out using recombinant NMDARs containing a GluN2A-N447K mutant that is located in the ligand-binding domain of the GluN2A subunit. Whole-cell current recordings in HEK 293T cells revealed that the N447K mutation increased the NMDAR current density by ~1.2-fold, enhanced the glutamate potency by 2-fold, and reduced the sensitivity to Mg2+ inhibition. These results indicated that N447K is a gain-of-function mutation. Interestingly, alternative substitutions by alanine and glutamic acid at the same residue (N447A and N447E) did not change NMDAR function, suggesting a residual dependence of this mutation in altering NMDAR function. Taken together, this study identified human GluN2A N447K as a novel mutation associated with epilepsy and validated its functional consequences in vitro. Identification of this mutation is also helpful for advancing our understanding of the role of NMDARs in epilepsy and provides new insights for precision therapeutics in epilepsy.
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References
Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, et al. Glutamate receptor ion channels: Structure, regulation, and function. Pharmacol Rev 2010, 62: 405–496.
Paoletti P, Bellone C, Zhou Q. NMDA receptor subunit diversity: Impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci 2013, 14: 383–400.
Burnashev N, Szepetowski P. NMDA receptor subunit mutations in neurodevelopmental disorders. Curr Opin Pharmacol 2015, 20: 73–82.
Myers CT, Mefford HC. Advancing epilepsy genetics in the genomic era. Genome Med 2015, 7: 91.
Yuan H, Low CM, Moody OA, Jenkins A, Traynelis SF. Ionotropic GABA and glutamate receptor mutations and human neurologic diseases. Mol Pharmacol 2015, 88: 203–217.
Wei F, Yan LM, Su T, He N, Lin ZJ, Wang J, et al. Ion channel genes and epilepsy: functional alteration, pathogenic potential, and mechanism of epilepsy. Neurosci Bull 2017, 33: 455–477.
Lemke JR, Geider K, Helbig KL, Heyne HO, Schutz H, Hentschel J, et al. Delineating the GRIN1 phenotypic spectrum: A distinct genetic NMDA receptor encephalopathy. Neurology 2016, 86: 2171–2178.
Hamdan FF, Gauthier J, Araki Y, Lin DT, Yoshizawa Y, Higashi K, et al. Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability. Am J Hum Genet 2011, 88: 306–316.
Ohba C, Shiina M, Tohyama J, Haginoya K, Lerman-Sagie T, Okamoto N, et al. GRIN1 mutations cause encephalopathy with infantile-onset epilepsy, and hyperkinetic and stereotyped movement disorders. Epilepsia 2015, 56: 841–848.
Endele S, Rosenberger G, Geider K, Popp B, Tamer C, Stefanova I, et al. Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes. Nat Genet 2010, 42: 1021–1026.
Yuan H, Hansen KB, Zhang J, Pierson TM, Markello TC, Fajardo KV, et al. Functional analysis of a de novo GRIN2A missense mutation associated with early-onset epileptic encephalopathy. Nat Commun 2014, 5: 3251.
Lemke JR, Lal D, Reinthaler EM, Steiner I, Nothnagel M, Alber M, et al. Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes. Nat Genet 2013, 45: 1067–1072.
Addis L, Virdee JK, Vidler LR, Collier DA, Pal DK, Ursu D. Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency–molecular profiling and functional rescue. Sci Rep 2017, 7: 66.
Chen W, Tankovic A, Burger PB, Kusumoto H, Traynelis SF, Yuan H. Functional evaluation of a de novo GRIN2A mutation identified in a patient with profound global developmental delay and refractory epilepsy. Mol Pharmacol 2017, 91: 317–330.
Lesca G, Rudolf G, Bruneau N, Lozovaya N, Labalme A, Boutry-Kryza N, et al. GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat Genet 2013, 45: 1061–1066.
Carvill GL, Regan BM, Yendle SC, O’Roak BJ, Lozovaya N, Bruneau N, et al. GRIN2A mutations cause epilepsy-aphasia spectrum disorders. Nat Genet 2013, 45: 1073–1076.
Gao K, Tankovic A, Zhang Y, Kusumoto H, Zhang J, Chen W, et al. A de novo loss-of-function GRIN2A mutation associated with childhood focal epilepsy and acquired epileptic aphasia. PLoS One 2017, 12: e0170818.
Lemke JR, Hendrickx R, Geider K, Laube B, Schwake M, Harvey RJ, et al. GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy. Ann Neurol 2014, 75: 147–154.
Swanger SA, Chen W, Wells G, Burger PB, Tankovic A, Bhattacharya S, et al. Mechanistic insight into NMDA receptor dysregulation by rare variants in the GluN2A and GluN2B agonist binding domains. Am J Hum Genet 2016, 99: 1261–1280.
Li D, Yuan H, Ortiz-Gonzalez XR, Marsh ED, Tian L, McCormick EM, et al. GRIN2D recurrent de novo dominant mutation causes a severe epileptic encephalopathy treatable with NMDA receptor channel blockers. Am J Hum Genet 2016, 99: 802–816.
Yi F, Mou TC, Dorsett KN, Volkmann RA, Menniti FS, Sprang SR, et al. Structural basis for negative allosteric modulation of GluN2A-containing NMDA receptors. Neuron 2016, 91: 1316–1329.
Yang W, Jiang LH. Site-directed mutagenesis to study the structure-function relationships of ion channels. Methods Mol Biol 2013, 998: 257–266.
Luo JH, Fu ZY, Losi G, Kim BG, Prybylowski K, Vissel B, et al. Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture. Neuropharmacology 2002, 42: 306–318.
Chen Q, He S, Hu XL, Yu J, Zhou Y, Zheng J, et al. Differential roles of NR2A- and NR2B-containing NMDA receptors in activity-dependent brain-derived neurotrophic factor gene regulation and limbic epileptogenesis. J Neurosci 2007, 27: 542–552.
Traynelis SF, Burgess MF, Zheng F, Lyuboslavsky P, Powers JL. Control of voltage-independent zinc inhibition of NMDA receptors by the NR1 subunit. J Neurosci 1998, 18: 6163–6175.
Mayer ML, Westbrook GL, Guthrie PB. Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 1984, 309: 261–263.
Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature 1984, 307: 462–465.
Paoletti P, Ascher P, Neyton J. High-affinity zinc inhibition of NMDA NR1-NR2A receptors. J Neurosci 1997, 17: 5711–5725.
Erreger K, Traynelis SF. Zinc inhibition of rat NR1/NR2A N-methyl-D-aspartate receptors. J Physiol 2008, 586: 763–778.
Hildebrand MS, Dahl HH, Damiano JA, Smith RJ, Scheffer IE, Berkovic SF. Recent advances in the molecular genetics of epilepsy. J Med Genet 2013, 50: 271–279.
Pierson TM, Yuan H, Marsh ED, Fuentes-Fajardo K, Adams DR, Markello T, et al. GRIN2A mutation and early-onset epileptic encephalopathy: Personalized therapy with memantine. Ann Clin Transl Neurol 2014, 1: 190–198.
Kearney JA. Precision medicine: NMDA receptor-targeted therapy for GRIN2D encephalopathy. Epilepsy Curr 2017, 17: 112–114.
de Ligt J, Willemsen MH, van Bon BW, Kleefstra T, Yntema HG, Kroes T, et al. Diagnostic exome sequencing in persons with severe intellectual disability. N Engl J Med 2012, 367: 1921–1929.
Ogden KK, Chen W, Swanger SA, McDaniel MJ, Fan LZ, Hu C, et al. Molecular mechanism of disease-associated mutations in the Pre-M1 helix of NMDA receptors and potential rescue pharmacology. PLoS Genet 2017, 13: e1006536.
Venkateswaran S, Myers KA, Smith AC, Beaulieu CL, Schwartzentruber JA, Consortium FC, et al. Whole-exome sequencing in an individual with severe global developmental delay and intractable epilepsy identifies a novel, de novo GRIN2A mutation. Epilepsia 2014, 55: e75–79.
Acknowledgements
This work was supported by the grants of the National Natural Science Foundation of China (81671162, 81521062, 81561168, and 81571273), the National Basic Research Development Program of China (2014CB910300 and 2013CB530904), and Key Research Project of the Ministry of Science and Technology of China (2016YFC0904400).
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Xu, XX., Liu, XR., Fan, CY. et al. Functional Investigation of a GRIN2A Variant Associated with Rolandic Epilepsy. Neurosci. Bull. 34, 237–246 (2018). https://doi.org/10.1007/s12264-017-0182-6
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DOI: https://doi.org/10.1007/s12264-017-0182-6