title: Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology.
abstract: N-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Kevin K
authors.identifier: http://orcid.org/0000-0001-7286-3209
authors.initials: KK
authors.lastname: Ogden
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Department of Neurology, Xiangya Hospital, Central South University, Changsha, China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Sharon A
authors.identifier: http://orcid.org/0000-0003-0615-6244
authors.initials: SA
authors.lastname: Swanger
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Miranda J
authors.identifier: http://orcid.org/0000-0003-4528-7108
authors.initials: MJ
authors.lastname: McDaniel
authors.affiliation: Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Linlin Z
authors.initials: LZ
authors.lastname: Fan
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Chun
authors.identifier: http://orcid.org/0000-0002-0949-9878
authors.initials: C
authors.lastname: Hu
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Anel
authors.identifier: http://orcid.org/0000-0002-0124-9495
authors.initials: A
authors.lastname: Tankovic
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Hirofumi
authors.identifier: http://orcid.org/0000-0001-7077-603X
authors.initials: H
authors.lastname: Kusumoto
authors.affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine and Pittsburgh Institute for Neurodegenerative Diseases, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Gabrielle J
authors.initials: GJ
authors.lastname: Kosobucki
authors.affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine and Pittsburgh Institute for Neurodegenerative Diseases, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Schulien
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Zhuocheng
authors.initials: Z
authors.lastname: Su
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Joseph
authors.identifier: http://orcid.org/0000-0003-2553-7018
authors.initials: J
authors.lastname: Pecha
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Subhrajit
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: Department of Medicine, The University of Melbourne, Austin Health and Royal Melbourne Hospital, Melbourne, Victoria, Australia.
authors.forename: Slavé
authors.initials: S
authors.lastname: Petrovski
authors.affiliation: Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, United States of America.
authors.forename: Adam E
authors.identifier: http://orcid.org/0000-0002-8699-2404
authors.initials: AE
authors.lastname: Cohen
authors.affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine and Pittsburgh Institute for Neurodegenerative Diseases, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Elias
authors.initials: E
authors.lastname: Aizenman
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Center for Functional Evaluation of Rare Variants (CFERV), Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia, United States of America.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Traynelis
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States of America.|Center for Functional Evaluation of Rare Variants (CFERV), Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia, United States of America.
authors.forename: Hongjie
authors.identifier: http://orcid.org/0000-0002-8127-7145
authors.initials: H
authors.lastname: Yuan
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008559:Memantine / Q000494:pharmacology; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009422:Nervous System Diseases / Q000235:genetics*; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D000072417:Protein Domains; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D018691:Excitatory Amino Acid Antagonists; D018698:Glutamic Acid; D008559:Memantine; D009419:Nerve Tissue Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C485668:GRIN1 protein, human; C121001:NR2B NMDA receptor