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.
Highlights
Recent analysis of whole exome data has shown that genes encoding excitatory post synaptic receptors, including the GRIN family, are some of the least tolerant genes in the body [1]
The increased use of next-generation sequencing for neurological patients has led to a growing catalog of patient-ascertained variants in N-methyl-D-aspartate receptor (NMDAR) subunits, which play important roles in normal brain development and have been implicated in epilepsy, language disorders, motor disorders, learning disorders, autism, attention deficit hyperactivity disorder, developmental delay, and schizophrenia
We use the largest currently available sample of human standing variation to illustrate the landscape of missense intolerance within the GluN1, GluN2A and GluN2B subunits, and provide the first evaluation of the molecular mechanisms of mutations in N-methyl-D-aspartate receptors (NMDARs) pre-M1 helix that links the agonist binding domain to the channel pore in patients with epilepsy and/or intellectual disability
Summary
Recent analysis of whole exome data has shown that genes encoding excitatory post synaptic receptors, including the GRIN family, are some of the least tolerant genes in the body [1]. They show significantly less non-synonymous variation than expected in specific regions [2], and harbor a large number of disease-associated de novo mutations ([3] [4]). Crystal structures of isolated ABDs of glutamate receptor ion channels revealed that upon agonist carboxy-terminal domain (in PINK).
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