Surface Expression, Function, and Pharmacology of Disease-Associated Mutations in the Membrane Domain of the Human GluN2B Subunit

Vyklický, Vojtěch; Krausová, Barbora; Černý, Jiří; Ladislav, Marek; Smejkalová, Tereza; Kysilov, Bohdan; Kořı́nek, Miloslav; Danačíková, Šárka; Hořák, Martin; Chodounská, Hana; Kudová, Eva; Vyklický, Ladislav

doi:10.3389/fnmol.2018.00110

Cited by 49 publications

(51 citation statements)

References 81 publications

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“…Recent genetic analysis of de novo mutations has identified additional ASD-associated mutations in GluN2B. Consistent with the observations reported here, several of these mutations produce NMDA receptors with reduced or abolished channel properties or trafficking to the cell surface in heterologous expression systems (Adams et al, 2014;Fedele et al, 2018;Liu et al, 2017;Swanger et al, 2016;Vyklicky et al, 2018). It will be important in future studies to compare the effects of distinct ASD mutations on NMDAR trafficking and function within neurons and to determine whether abnormal dendrite development is a common phenotypic outcome of ASD-associated GluN2B mutations.…”

Section: Discussionsupporting

confidence: 87%

“…The recurrence of de novo ASD-associated mutations in GRIN2B and rigorous analyses indicate that GRIN2B is a true ASD-associated gene (De Rubeis et al, 2014;Iossifov et al, 2014;O'Roak et al, 2012b;Stessman et al, 2017). However, we are only beginning to understand the impact of ASD-associated GRIN2B mutations on NMDAR function (Fedele et al, 2018;Liu et al, 2017;Vyklicky et al, 2018), and it is not yet clear how GRIN2B mutations alter neuronal development to cause ASD.…”

Section: Introductionmentioning

confidence: 99%

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An autism-associated mutation in GluN2B prevents NMDA receptor trafficking and interferes with dendrite growth

Sceniak

Fedder

Wang

et al. 2019

Journal of Cell Science

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Autism spectrum disorders (ASDs) are neurodevelopmental disorders with multiple genetic associations. Analysis of de novo mutations identified GRIN2B, which encodes the GluN2B subunit of NMDA receptors, as a gene linked to ASDs with high probability. However, the mechanisms by which GRIN2B mutations contribute to ASD pathophysiology are not understood. Here, we investigated the cellular phenotypes induced by a human mutation that is predicted to truncate GluN2B within the extracellular loop. This mutation abolished NMDA-dependent Ca 2+ influx. Mutant GluN2B co-assembled with GluN1 but was not trafficked to the cell surface or dendrites. When mutant GluN2B was expressed in developing cortical neurons, dendrites appeared underdeveloped, with shorter and fewer branches, while spine density was unaffected. Mutant dendritic arbors were often dysmorphic, displaying abnormal filopodial-like structures. Interestingly, dendrite maldevelopment appeared when mutant GluN2B was expressed on a wild-type background, reflecting the disease given that individuals are heterozygous for GRIN2B mutations. Restoring the fourth transmembrane domain and cytoplasmic tail did not rescue the phenotypes. Finally, abnormal development was not accompanied by reduced mTOR signaling. These data suggest that mutations in GluN2B contribute to ASD pathogenesis by disrupting dendrite development.

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Section: Discussionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

An autism-associated mutation in GluN2B prevents NMDA receptor trafficking and interferes with dendrite growth

Sceniak

Fedder

Wang

et al. 2019

Journal of Cell Science

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“…For this study, we used cDNA vectors expressing untagged human GluN1-4a (hGluN1-4a; NCBI RefSeq NM_001270610.1 was modified to the human version by changing the four amino acid residues (N159S, R212K, I267L, M415L) that differ between the rat and human GluN1-4a subunits) 27 , GluN2A (hGluN2A; NCBI Reference Sequence: NM_000833.5), GluN2B (hGluN2B; NCBI RefSeq: NM_000834.5), and GluN3A (hGluN3A; NCBI RefSeq: NM_133445.3) subunits 27 , 75 , 76 , GFP-tagged versions of rat GluN2A (GFP-rGluN2A; NCBI RefSeq: NM_012573.3) and GluN2B (GFP-rGluN2B; NCBI RefSeq: NM_012574.1) subunits, and GFP-tagged human GluN3A (GFP-hGluN3A; NCBI RefSeq: NM_133445.3) 27 , 77 . The YFP-tagged hGluN1-1a subunit (NCBI RefSeq NM_017010.2 was modified to the human version as described above) 76 was cloned into the FHUGW lentivirus vector containing 20 sense nucleotides in the GluN1 target sequence (gac cgg aag ttt gcc aac ta; with a short hairpin (AAGCTT) and 20 antisense nucleotides cloned downstream of the H1 promoter) to knock down the endogenous GluN1 subunit, as described previously 53 . Silent mutations (gac cgC aaA ttC gcG aac ta; the mutated nucleotides are indicated in capital letters) were introduced in order to generate shRNA-resistant versions of YFP-hGluN1-1a and YFP-hGluN1-1aS688Y .…”

Section: Methodsmentioning

confidence: 99%

The pathogenic S688Y mutation in the ligand-binding domain of the GluN1 subunit regulates the properties of NMDA receptors

Skřenková

Song

Kortus

et al. 2020

Sci Rep

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Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and d-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.

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“…Our experiments highlight the critical role the LBD-TMD linkers play in regulating NMDAR activity. Notable are the S2-M4/M4 segments which display numerous disease-associated variants (Yuan et al, 2014;Amin et al, 2018;Vyklicky et al, 2018;Amin et al, 2020). Further, the extracellular position of S2-M4 compared to the membrane-embedded position of M4 provides a strategic advantage for designing new drug therapies (Shi et al, 2019).…”

Section: Resultsmentioning

confidence: 99%

NMDA receptors require multiple pre-opening gating steps for efficient synaptic activity

Amin

Gochman

et al. 2020

Preprint

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NMDA receptors (NMDAR) are glutamate-gated ion channels that mediate the majority of fast excitatory synaptic transmission in the nervous system. A central feature of NMDAR physiology is the opening of the ion channel driven by presynaptically-released glutamate. Using glutamate applications to outside-out patches containing a single NMDAR in the continuous presence of the co-agonist glycine, we find that agonist-bound receptors transition to the open state via two conformations, an 'unconstrained pre-active' state that can rapidly transition to the open state and contributes to synaptic events, and a 'constrained pre-active' state that requires more energy and hence time to open and does not contribute to fast signaling. To define how agonist binding might drive these conformations, we decoupled the ligand-binding domains from specific transmembrane segments for the GluN1 and GluN2A subunits. Displacements of the central pore-forming M3 segments define the energy of fast channel opening. However, to enter the unconstrained conformation and contribute to fast signaling, a peripheral helix, the GluN2 pre-M1, must be displaced before the M3 segments move. This pre-M1 displacement is facilitated by the flexibility of another nearby peripheral element, the GluN1 and GluN2A S2-M4. We conclude that peripheral structural elements -pre-M1 and S2-M4 -work in concert to remove constraints and prime the channel for rapid opening, thus facilitating fast synaptic transmission. 214 words. McDaniel et al., 2020). The S2-M4 linker/M4 segment also regulates gating (Amin et al., 2017;Yelshanskaya et al., 2017;Shi et al., 2019). Still, the energetics and timing of these non-pore forming elements to receptor gating remains incomplete.Here, we address the gating mechanism in NMDARs. At most synapses, NMDARs are obligate hetero-tetramers composed of two GluN1 and two GluN2 (A-D) subunits. To define conformational changes between agonist binding and pore opening, we rapidly applied glutamate to outside-out patches containing single NMDARs and assessed the time between when glutamate was first applied to when the channel first opened ('latency to 1 st opening', see Materials and Methods) (Aldrich et al., 1983). With this approach, we identified that wild type GluN1/GluN2A NMDARs, in the transition from agonist-bound closed to the open state, transitions through one of two pre-active states: an 'unconstrained' state that can rapidly transition to the open state and contributes to fast synaptic signaling, and a 'constrained' state that slowly transitions to the open state, presumably due to higher energy requirements, and does not contribute to fast signaling. Further, by decoupling the LBD from the TMD for each specific LBD-TMD linker (S1-M1, M3-S2, and S2-M4, Figure 1B & 1C) (Niu et al., 2004;Kazi et al., 2014), we show that the non-pore forming linkers uniquely influence pore-opening by altering the stability of these pre-active conformations. Thus, our results highlight the temporal and coordinated movements from agonist binding to io...

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Surface Expression, Function, and Pharmacology of Disease-Associated Mutations in the Membrane Domain of the Human GluN2B Subunit

Cited by 49 publications

References 81 publications

An autism-associated mutation in GluN2B prevents NMDA receptor trafficking and interferes with dendrite growth

An autism-associated mutation in GluN2B prevents NMDA receptor trafficking and interferes with dendrite growth

The pathogenic S688Y mutation in the ligand-binding domain of the GluN1 subunit regulates the properties of NMDA receptors

NMDA receptors require multiple pre-opening gating steps for efficient synaptic activity

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