Disease-associated missense mutations in GluN2B subunit alter NMDA receptor ligand binding and ion channel properties

Laura Fedele,Trevor G Smart,Maya Topf,Alasdair Gibb,Joseph Newcombe,Robert J Harvey

doi:10.1038/s41467-018-02927-4

Abstract

Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes. Here, we investigated several such mutations using a near-complete, hybrid 3D model of the human NMDAR and studied their consequences with kinetic modelling and electrophysiology. The mutants revealed reductions in glutamate potency; increased receptor desensitisation; and ablation of voltage-dependent Mg2+ block. In addition, we provide new views on Mg2+ and NMDA channel blocker binding sites. We demonstrate that these mutants have significant impact on excitatory transmission in developing neurons, revealing profound changes that could underlie their associated neurological disorders. Of note, the NMDAR channel mutant GluN2BV618G unusually allowed Mg2+ permeation, whereas nearby N615I reduced Ca2+ permeability. By identifying the binding site for an NMDAR antagonist that is used in the clinic to rescue gain-of-function phenotypes, we show that drug binding may be modified by some GluN2B disease-causing mutations.

Highlights

Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes
To understand the consequences of NMDAR mutations on neuronal activity, here we have studied a range of de novo missense mutations affecting the GluN2B subunit, subsequently profiling four in detail, C461F, P553L, N615I and V618G, which are associated with neurodevelopmental disorders in children[5,6,9]
C461F features in an individual with Lennox Gastaut syndrome with autistic features[5]; P553L was present in another subject with severe intellectual disability[9]; and N615I and V618G both associate with West syndrome[6]

Summary

Introduction

Genetic and bioinformatic analyses have identified missense mutations in GRIN2B encoding the NMDA receptor GluN2B subunit in autism, intellectual disability, Lennox Gastaut and West Syndromes. To understand the consequences of NMDAR mutations on neuronal activity, here we have studied a range of de novo missense mutations affecting the GluN2B subunit, subsequently profiling four in detail, C461F, P553L, N615I and V618G, which are associated with neurodevelopmental disorders in children[5,6,9] These mutations were selected because of bioinformatic predictions of pathogenicity, and because they are structurally diverse, involving functionally important domains in NMDARs. In addition, we wanted to explore potential links between NMDAR dysfunction and clinical phenotypes. C461F features in an individual with Lennox Gastaut syndrome with autistic features[5]; P553L was present in another subject with severe intellectual disability[9]; and N615I and V618G both associate with West syndrome[6] We investigated how these mutations affected the structure and function of NMDARs in vitro before examining how excitatory transmission was perturbed in situ. Our hybrid NMDAR model closely aligns with the pore region of the Xenopus laevis structure (PDB 4TLM; Supplementary Fig. 1) that is considered to reside in a trapped conformation following co-crystallisation with the NMDA channel blocker, MK-80110

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