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- PDB-7sk2: Human wildtype GABA reuptake transporter 1 in complex with tiagab... -

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Basic information

Entry
Database: PDB / ID: 7sk2
TitleHuman wildtype GABA reuptake transporter 1 in complex with tiagabine, inward-open conformation
ComponentsSodium- and chloride-dependent GABA transporter 1
KeywordsMEMBRANE PROTEIN / Neurotransmitter transporter
Function / homology
Function and homology information


gamma-aminobutyric acid reuptake / Reuptake of GABA / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to sucrose ...gamma-aminobutyric acid reuptake / Reuptake of GABA / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to sucrose / response to purine-containing compound / Na+/Cl- dependent neurotransmitter transporters / sodium ion import across plasma membrane / amino acid transport / associative learning / transport across blood-brain barrier / sodium ion transmembrane transport / : / GABA-ergic synapse / chloride transmembrane transport / response to cocaine / response to lead ion / synapse organization / response to toxic substance / memory / response to calcium ion / response to estradiol / presynaptic membrane / chemical synaptic transmission / postsynaptic membrane / axon / neuronal cell body / cell surface / membrane / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, GABA, GAT-1 / Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile.
Similarity search - Domain/homology
Tiagabine / Sodium- and chloride-dependent GABA transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.82 Å
AuthorsGati, C. / Motiwala, Z. / Aduri, N.G. / Shaye, H. / Han, G.W. / Cherezov, V.
Funding support1items
OrganizationGrant numberCountry
Other private
CitationJournal: Nature / Year: 2022
Title: Structural basis of GABA reuptake inhibition.
Authors: Zenia Motiwala / Nanda Gowtham Aduri / Hamidreza Shaye / Gye Won Han / Jordy Homing Lam / Vsevolod Katritch / Vadim Cherezov / Cornelius Gati /
Abstract: γ-Aminobutyric acid (GABA) transporter 1 (GAT1) regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release ...γ-Aminobutyric acid (GABA) transporter 1 (GAT1) regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.
History
DepositionOct 19, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 6, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium- and chloride-dependent GABA transporter 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,2622
Polymers64,8871
Non-polymers3761
Water00
1


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

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Components

#1: Protein Sodium- and chloride-dependent GABA transporter 1 / GAT-1 / Solute carrier family 6 member 1


Mass: 64886.793 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A1, GABATR, GABT1, GAT1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P30531
#2: Chemical ChemComp-TGI / Tiagabine / (3R)-1-[4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl]piperidine-3-carboxylic acid / Gabitril


Mass: 375.548 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H25NO2S2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
Has ligand of interestY

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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: GAT-1 in complex with tiagabine / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 67 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2150 mMsodium chlorideNaCl1
30.001 %detergentLMNG1
SpecimenConc.: 18 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 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 / Calibrated magnification: 105000 X / Nominal defocus max: 25000 nm / Nominal defocus min: 10000 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: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 39572
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18_3861: / Classification: refinement
EM software
IDNameVersionCategory
2EPU2image acquisition
4cryoSPARCv3.2.0+210831CTF correction
10cryoSPARCv3.2.0+210831initial Euler assignment
11cryoSPARCv3.2.0+210831final Euler assignment
13cryoSPARCv3.2.0+2108313D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135297 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044313
ELECTRON MICROSCOPYf_angle_d1.0115883
ELECTRON MICROSCOPYf_dihedral_angle_d8.945582
ELECTRON MICROSCOPYf_chiral_restr0.055642
ELECTRON MICROSCOPYf_plane_restr0.006722

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