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- PDB-8fqb: GluA2 flip Q isoform of AMPA receptor in complex with gain-of-fun... -

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Basic information

Entry
Database: PDB / ID: 8fqb
TitleGluA2 flip Q isoform of AMPA receptor in complex with gain-of-function TARP gamma2, with 10mM CaCl2, 140mM NMDG, 330uM CTZ, and 100mM L-glutamate (Open-Ca10)
Components
  • Glutamate receptor 2
  • Voltage-dependent calcium channel gamma-2 subunit
KeywordsTRANSPORT PROTEIN / Inotropic glutamate receptors / AMPA receptors / ligand gated ion channel / auxiliary subunit / TARP / stargazin / TARP gamma2 / glutamate / calcium / neurotransmitter receptor / synaptic transmission
Function / homology
Function and homology information


Presynaptic depolarization and calcium channel opening / LGI-ADAM interactions / Trafficking of AMPA receptors / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / regulation of AMPA receptor activity / membrane hyperpolarization / nervous system process ...Presynaptic depolarization and calcium channel opening / LGI-ADAM interactions / Trafficking of AMPA receptors / eye blink reflex / positive regulation of protein localization to basolateral plasma membrane / cerebellar mossy fiber / postsynaptic neurotransmitter receptor diffusion trapping / 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 head / cellular response to amine stimulus / neurotransmitter receptor localization to postsynaptic specialization membrane / neuromuscular junction development / perisynaptic space / Activation of AMPA receptors / ligand-gated monoatomic cation channel activity / AMPA glutamate receptor activity / transmission of nerve impulse / response to lithium ion / Trafficking of GluR2-containing AMPA receptors / kainate selective glutamate receptor activity / AMPA glutamate receptor complex / cellular response to glycine / extracellularly glutamate-gated ion channel activity / immunoglobulin binding / asymmetric synapse / ionotropic glutamate receptor complex / conditioned place preference / regulation of receptor recycling / membrane depolarization / glutamate receptor binding / Unblocking of NMDA receptors, glutamate binding and activation / positive regulation of synaptic transmission / regulation of postsynaptic membrane neurotransmitter receptor levels / regulation of synaptic transmission, glutamatergic / voltage-gated calcium channel activity / response to fungicide / cytoskeletal protein binding / glutamate-gated receptor activity / regulation of long-term synaptic depression / extracellular ligand-gated monoatomic ion channel activity / cellular response to brain-derived neurotrophic factor stimulus / presynaptic active zone membrane / glutamate-gated calcium ion channel activity / somatodendritic compartment / dendrite membrane / 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 / hippocampal mossy fiber to CA3 synapse / dendritic shaft / SNARE binding / regulation of membrane potential / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic transmission, glutamatergic / PDZ domain binding / protein tetramerization / establishment of protein localization / response to calcium ion / postsynaptic density membrane / cerebral cortex development / modulation of chemical synaptic transmission / receptor internalization / Schaffer collateral - CA1 synapse / terminal bouton / synaptic vesicle / synaptic vesicle membrane / presynapse / signaling receptor activity / amyloid-beta binding / presynaptic membrane / growth cone / scaffold protein binding / perikaryon / chemical synaptic transmission / dendritic spine / postsynaptic membrane / neuron projection / postsynaptic density / axon / external side of plasma membrane / neuronal cell body / dendrite / synapse / protein kinase binding / protein-containing complex binding / glutamatergic synapse / cell surface / endoplasmic reticulum / protein-containing complex / identical protein binding / membrane / plasma membrane
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
Voltage-dependent calcium channel gamma-2 subunit / Glutamate receptor 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.36 Å
AuthorsNakagawa, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH123474 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: The open gate of the AMPA receptor forms a Ca binding site critical in regulating ion transport.
Authors: Terunaga Nakagawa / Xin-Tong Wang / Federico J Miguez-Cabello / Derek Bowie /
Abstract: Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating ...Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating mechanism has been studied extensively, understanding how cations traverse the pore has remained elusive. Here we investigated putative ion and water densities in the open pore of Ca-permeable AMPARs (rat GRIA2 flip-Q isoform) at 2.3-2.6 Å resolution. We show that the ion permeation pathway attains an extracellular Ca binding site (site-G) when the channel gate moves into the open configuration. Site-G is highly selective for Ca over Na, favoring the movement of Ca into the selectivity filter of the pore. Seizure-related N619K mutation, adjacent to site-G, promotes channel opening but attenuates Ca binding and thus diminishes Ca permeability. Our work identifies the importance of site-G, which coordinates with the Q/R site of the selectivity filter to ensure the preferential transport of Ca through the channel pore.
History
DepositionJan 5, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2May 1, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate receptor 2
B: Glutamate receptor 2
C: Glutamate receptor 2
D: Glutamate receptor 2
E: Voltage-dependent calcium channel gamma-2 subunit
G: Voltage-dependent calcium channel gamma-2 subunit
F: Voltage-dependent calcium channel gamma-2 subunit
H: Voltage-dependent calcium channel gamma-2 subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)546,78811
Polymers546,6778
Non-polymers1113
Water3,171176
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Glutamate receptor 2 / GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / ...GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / AMPA 2 / GluA2


Mass: 99559.461 Da / Num. of mol.: 4
Fragment: DYKDDDDK near the C-terminal is a FLAG epitope tag used for purification
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Cell line (production host): HEK / Production host: Homo sapiens (human) / References: UniProt: P19491
#2: Protein
Voltage-dependent calcium channel gamma-2 subunit / Neuronal voltage-gated calcium channel gamma-2 subunit / Stargazin / Transmembrane AMPAR regulatory ...Neuronal voltage-gated calcium channel gamma-2 subunit / Stargazin / Transmembrane AMPAR regulatory protein gamma-2 / TARP gamma-2


Mass: 37109.809 Da / Num. of mol.: 4 / Mutation: K52E, K53E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Cacng2, Stg / Cell line (production host): HEK / Production host: Homo sapiens (human) / References: UniProt: O88602
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 176 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Homotetrameric assembly of GluA2 flip-Q isoform in complex with TARP gamma2 (K52E, K53E). Dimer-of-dimers assembly.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK
Buffer solutionpH: 8
Details: L-glutamic acid (100mM) and cyclothiazide (CTZ, 0.33mM) was added before freezing. The 1M L-glutamic acid stock solution is adjusted to pH 7.4 using NaOH.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris bufferTrisHCl1
210 mMCalcium ChlorideCaCl21
3140 mMN-methyl-D-glucamineNMDG1
40.03 %GDNGDN1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 28505

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 986075 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210910
ELECTRON MICROSCOPYf_angle_d0.43914776
ELECTRON MICROSCOPYf_dihedral_angle_d3.1621464
ELECTRON MICROSCOPYf_chiral_restr0.0341672
ELECTRON MICROSCOPYf_plane_restr0.0031804

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