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- EMDB-47793: Structure of AMPA receptor GluA2 and auxiliary subunit TARP gamma... -

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Entry
Database: EMDB / ID: EMD-47793
TitleStructure of AMPA receptor GluA2 and auxiliary subunit TARP gamma-2 (LBD-TMD) in complex with anti-miR 17 oligonucleotide RGLS4326
Map data
Sample
  • Complex: GluA2-y2 + oligo RGLS 4326
    • Protein or peptide: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
  • Ligand: O-{[(1S,3S,4R,6S,7R)-7-{[(R)-{[(1R,3R,4R,6S,7R)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-7-hydroxy-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methoxy}(sulfanyl)phosphoryl]oxy}-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methyl} O-[(2R,3S,4R,5S)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxy-2-methyloxolan-3-yl] hydrogen (R)-phosphorothioate
KeywordsAMPA receptor / TARP gamma-2 / ion channel / anti-miR 17 / oligonucleotide / SIGNALING PROTEIN
Function / homology
Function and homology information


Presynaptic depolarization and calcium channel opening / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / channel regulator activity / LGI-ADAM interactions / Trafficking of AMPA receptors / regulation of AMPA receptor activity / membrane hyperpolarization ...Presynaptic depolarization and calcium channel opening / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / channel regulator activity / LGI-ADAM interactions / Trafficking of AMPA receptors / regulation of AMPA receptor activity / membrane hyperpolarization / nervous system process / protein targeting to membrane / voltage-gated calcium channel complex / spine synapse / dendritic spine neck / dendritic spine cytoplasm / cellular response to amine stimulus / dendritic spine head / neurotransmitter receptor localization to postsynaptic specialization membrane / perisynaptic space / Activation of AMPA receptors / ligand-gated monoatomic cation channel activity / neuromuscular junction development / AMPA glutamate receptor activity / response to lithium ion / Trafficking of GluR2-containing AMPA receptors / transmission of nerve impulse / AMPA glutamate receptor clustering / kainate selective glutamate receptor activity / cellular response to glycine / AMPA glutamate receptor complex / immunoglobulin binding / asymmetric synapse / extracellularly glutamate-gated ion channel activity / regulation of receptor recycling / ionotropic glutamate receptor complex / membrane depolarization / conditioned place preference / glutamate receptor binding / Unblocking of NMDA receptors, glutamate binding and activation / positive regulation of synaptic transmission / positive regulation of synaptic transmission, glutamatergic / regulation of synaptic transmission, glutamatergic / regulation of postsynaptic membrane neurotransmitter receptor levels / response to fungicide / voltage-gated calcium channel activity / glutamate-gated receptor activity / cytoskeletal protein binding / extracellular ligand-gated monoatomic ion channel activity / regulation of long-term synaptic depression / cellular response to brain-derived neurotrophic factor stimulus / glutamate-gated calcium ion channel activity / presynaptic active zone membrane / somatodendritic compartment / dendrite membrane / excitatory synapse / ionotropic glutamate receptor binding / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / synaptic membrane / positive regulation of excitatory postsynaptic potential / hippocampal mossy fiber to CA3 synapse / dendritic shaft / SNARE binding / PDZ domain binding / calcium channel regulator activity / synaptic transmission, glutamatergic / regulation of membrane potential / protein tetramerization / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / establishment of protein localization / response to calcium ion / cerebral cortex development / postsynaptic density membrane / modulation of chemical synaptic transmission / receptor internalization / Schaffer collateral - CA1 synapse / terminal bouton / synaptic vesicle / long-term synaptic potentiation / synaptic vesicle membrane / signaling receptor activity / amyloid-beta binding / presynapse / growth cone / presynaptic membrane / scaffold protein binding / dendritic spine / chemical synaptic transmission / perikaryon / postsynaptic membrane / neuron projection / postsynaptic density / axon / external side of plasma membrane / neuronal cell body / synapse / dendrite / protein kinase binding
Similarity search - Function
Voltage-dependent calcium channel, gamma-2 subunit / : / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel ...Voltage-dependent calcium channel, gamma-2 subunit / : / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / : / Ionotropic glutamate receptor / Eukaryotic homologues of bacterial periplasmic substrate binding proteins. / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
Glutamate receptor 2 / Voltage-dependent calcium channel gamma-2 subunit
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsYen LY / Gangwar SP / Yelshanskaya MV / Sobolevsky AI
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS083660 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS107253 United States
CitationJournal: Nat Commun / Year: 2025
Title: The nucleobase guanine at the 3'-terminus of oligonucleotide RGLS4326 drives off-target AMPAR inhibition and CNS toxicity.
Authors: Tania Valencia / Laura Y Yen / Cindy Berman / Thomas Vincent / Scott Davis / Francesca Varrone / Jianfeng Huang / Jessica Mastroianni / Morgan Carlson / Tate Owen / Amin Kamel / Denis Drygin ...Authors: Tania Valencia / Laura Y Yen / Cindy Berman / Thomas Vincent / Scott Davis / Francesca Varrone / Jianfeng Huang / Jessica Mastroianni / Morgan Carlson / Tate Owen / Amin Kamel / Denis Drygin / Garth A Kinberger / Shanti Pal Gangwar / Maria V Yelshanskaya / John Ridley / Robert Kirby / Jesus Alvarez / Ronak Lakhia / Vishal Patel / Alexander I Sobolevsky / Edmund C Lee /
Abstract: Designing safe and effective oligonucleotide (ON) therapeutics requires thorough understanding of structural-activity relationship (SAR) with the intended on-target(s) as well as the unintended off- ...Designing safe and effective oligonucleotide (ON) therapeutics requires thorough understanding of structural-activity relationship (SAR) with the intended on-target(s) as well as the unintended off-target(s). Despite encouraging pharmacodynamic activity in a Phase 1b study, development of the first-generation anti-miR-17 ON RGLS4326 for the treatment of autosomal dominant polycystic kidney disease was discontinued due to dose-limiting central nervous system (CNS)-related toxicity observed in nonclinical chronic toxicity studies. Here, we provide SAR evidence that the nucleobase guanine at the 3'-terminus of RGLS4326 drives an unexpected off-target aptamer-like direct interaction with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), thereby causing CNS toxicity. By replacing the 3'-terminal guanine with adenine, we discover the next-generation anti-miR-17 RGLS8429 that is devoid of off-target AMPAR interaction and CNS toxicity while preserving the potency against the on-target miR-17. Here, we show a way to avoid off-target CNS effects and, more importantly, data that support the clinical development of RGLS8429.
History
DepositionNov 8, 2024-
Header (metadata) releaseFeb 11, 2026-
Map releaseFeb 11, 2026-
UpdateFeb 11, 2026-
Current statusFeb 11, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47793.png

Downloads & links

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Map

FileDownload / File: emd_47793.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.19
Minimum - Maximum-1.6262423 - 2.3999536
Average (Standard dev.)0.0015574993 (±0.043481797)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 332.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_47793_half_map_1.map
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Half map: #1

Fileemd_47793_half_map_2.map
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Sample components

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Entire : GluA2-y2 + oligo RGLS 4326

EntireName: GluA2-y2 + oligo RGLS 4326
Components
  • Complex: GluA2-y2 + oligo RGLS 4326
    • Protein or peptide: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
  • Ligand: O-{[(1S,3S,4R,6S,7R)-7-{[(R)-{[(1R,3R,4R,6S,7R)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-7-hydroxy-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methoxy}(sulfanyl)phosphoryl]oxy}-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methyl} O-[(2R,3S,4R,5S)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxy-2-methyloxolan-3-yl] hydrogen (R)-phosphorothioate

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Supramolecule #1: GluA2-y2 + oligo RGLS 4326

SupramoleculeName: GluA2-y2 + oligo RGLS 4326 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: LBD-TMD of AMPA receptor GluA2 in complex with auxiliary subunit TARP gamma-2 bound to anti-miR 17 oligonucleotide RGLS 4326
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium ch...

MacromoleculeName: Isoform Flip of Glutamate receptor 2,Voltage-dependent calcium channel gamma-2 subunit
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 114.921297 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: NSIQIGGLFP RGADQEYSAF RVGMVQFSTS EFRLTPHIDN LEVANSFAVT NAFCSQFSRG VYAIFGFYDK KSVNTITSFC GTLHVSFIT PSFPTDGTHP FVIQMRPDLK GALLSLIEYY QWDKFAYLYD SDRGLSTLQA VLDSAAEKKW QVTAINVGNI N NDKKDETY ...String:
NSIQIGGLFP RGADQEYSAF RVGMVQFSTS EFRLTPHIDN LEVANSFAVT NAFCSQFSRG VYAIFGFYDK KSVNTITSFC GTLHVSFIT PSFPTDGTHP FVIQMRPDLK GALLSLIEYY QWDKFAYLYD SDRGLSTLQA VLDSAAEKKW QVTAINVGNI N NDKKDETY RSLFQDLELK KERRVILDCE RDKVNDIVDQ VITIGKHVKG YHYIIANLGF TDGDLLKIQF GGAEVSGFQI VD YDDSLVS KFIERWSTLE EKEYPGAHTA TIKYTSALTY DAVQVMTEAF RNLRKQRIEI SRRGNAGDCL ANPAVPWGQG VEI ERALKQ VQVEGLSGNI KFDQNGKRIN YTINIMELKT NGPRKIGYWS EVDKMVLTED DTSGLEQKTV VVTTILESPY VMMK KNHEM LEGNERYEGY CVDLAAEIAK HCGFKYKLTI VGDGKYGARD ADTKIWNGMV GELVYGKADI AIAPLTITLV REEVI DFSK PFMSLGISIM IKKPQKSKPG VFSFLDPLAY EIWMCIVFAY IGVSVVLFLV SRFSPYEWHT EEFEDGRETQ SSESTN EFG IFNSLWFSLG AFMQQGCDIS PRSLSGRIVG GVWWFFTLII ISSYTANLAA FLTVERMVSP IESAEDLSKQ TEIAYGT LD SGSTKEFFRR SKIAVFDKMW TYMRSAEPSV FVRTTAEGVA RVRKSKGKYA YLLESTMNEY IEQRKPCDTM KVGGNLDS K GYGIATPKGS SLGTPVNLAV LKLSEQGVLD KLKNKWWYDK GECGAKDSGS KEKTSALSLS NVAGVFYILV GGLGLAMLV ALIEFCYKSR AEAKRMKGTG LFDRGVQMLL TTVGAFAAFS LMTIAVGTDY WLYSRGVCKT KSVSEDETSK KNEEVMTHSG LWRTCCLEG NFKGLCKQID HFPEDADYEA DTAEYFLRAV RASSIFPILS VILLFMGGLC IAASEFYKTR HNIILSAGIF F VSAGLSNI IGIIVYISAN AGDPSKSDSK KNSYSYGWSF YFGALSFIIA EMVGVLAVHM FIDRHKQLTG

UniProtKB: Glutamate receptor 2, Voltage-dependent calcium channel gamma-2 subunit

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Macromolecule #2: O-{[(1S,3S,4R,6S,7R)-7-{[(R)-{[(1R,3R,4R,6S,7R)-3-(2-amino-6-oxo-...

MacromoleculeName: O-{[(1S,3S,4R,6S,7R)-7-{[(R)-{[(1R,3R,4R,6S,7R)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-7-hydroxy-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methoxy}(sulfanyl)phosphoryl]oxy}-3-(2,4-dioxo- ...Name: O-{[(1S,3S,4R,6S,7R)-7-{[(R)-{[(1R,3R,4R,6S,7R)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-7-hydroxy-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methoxy}(sulfanyl)phosphoryl]oxy}-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6-methyl-2,5-dioxabicyclo[2.2.1]heptan-1-yl]methyl} O-[(2R,3S,4R,5S)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxy-2-methyloxolan-3-yl] hydrogen (R)-phosphorothioate
type: ligand / ID: 2 / Number of copies: 4 / Formula: A1BX3
Molecular weightTheoretical: 977.806 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.24 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3tris
150.0 mMNaClsodium chloride
0.05 %C56H92O29digitonin
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsProtein extracted and purified in detergent micelle

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Initial model generated from the data (ab initio)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 192000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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