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- PDB-9r1h: Inward-occluded structure of human glycine transporter 2 bound to... -

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Basic information

Entry
Database: PDB / ID: 9r1h
TitleInward-occluded structure of human glycine transporter 2 bound to substrate glycine
ComponentsSodium- and chloride-dependent glycine transporter 2
KeywordsMEMBRANE PROTEIN / Transport protein / SLC6A5 / neurotransmitter/sodium symporter / Sodium- and chloride-dependent glycine transporter 2
Function / homology
Function and homology information


Defective SLC6A5 causes hyperekplexia 3 (HKPX3) / glycine:sodium symporter activity / synaptic transmission, glycinergic / glycine import across plasma membrane / dense core granule / SLC-mediated transport of neurotransmitters / neurotransmitter transport / sodium ion transmembrane transport / chemical synaptic transmission / endosome ...Defective SLC6A5 causes hyperekplexia 3 (HKPX3) / glycine:sodium symporter activity / synaptic transmission, glycinergic / glycine import across plasma membrane / dense core granule / SLC-mediated transport of neurotransmitters / neurotransmitter transport / sodium ion transmembrane transport / chemical synaptic transmission / endosome / synapse / metal ion binding / membrane / plasma membrane
Similarity search - Function
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
GLYCINE / Sodium- and chloride-dependent glycine transporter 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsCantwell Chater, R.P. / Peiser-Oliver, J. / Pati, T.K. / Quinn, A.S. / Lotsaris, I. / Frangos, Z.J. / Anderson, K.E. / Tischer, A.E. / Williams-Noonan, B.J. / Aubrey, K.R. ...Cantwell Chater, R.P. / Peiser-Oliver, J. / Pati, T.K. / Quinn, A.S. / Lotsaris, I. / Frangos, Z.J. / Anderson, K.E. / Tischer, A.E. / Williams-Noonan, B.J. / Aubrey, K.R. / O Mara, M.L. / Michaelides, M. / Mohammadi, S.A. / Cioffi, C.L. / Vandenberg, R.J. / Shahsavar, A.
Funding support Denmark, United States, 3items
OrganizationGrant numberCountry
LundbeckfondenR368-2021-522 Denmark
Novo Nordisk FoundationNNF23OC0087107 Denmark
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)R01DA048879 United States
CitationJournal: Nat Commun / Year: 2026
Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects.
Authors: Ryan P Cantwell Chater / Julian Peiser-Oliver / Tanmay K Pati / Ada S Quinn / Irina Lotsaris / Zachary J Frangos / Kristen E Anderson / Anna E Tischer / Billy J Williams-Noonan / Karin R ...Authors: Ryan P Cantwell Chater / Julian Peiser-Oliver / Tanmay K Pati / Ada S Quinn / Irina Lotsaris / Zachary J Frangos / Kristen E Anderson / Anna E Tischer / Billy J Williams-Noonan / Karin R Aubrey / Megan L O'Mara / Michael Michaelides / Sarasa A Mohammadi / Christopher L Cioffi / Robert J Vandenberg / Azadeh Shahsavar /
Abstract: Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, ...Chronic neuropathic pain, caused by nerve damage or disease, is increasing in prevalence, but current treatments are ineffective and over-reliant on opioids. The neuronal glycine transporter, GlyT2, regulates inhibitory glycinergic neurotransmission and represents a promising target for new analgesics. However, most GlyT2 inhibitors cause significant side effects, in part due to irreversible inhibition at analgesic doses. Here we develop a reversible inhibitor of GlyT2, RPI-GLYT2-82, and identify its binding site by determining cryo-EM structures of human GlyT2. We capture three fundamental conformational states of GlyT2 in the substrate-free state, and bound to either glycine, RPI-GLYT2-82 or the pseudo-irreversible inhibitor ORG25543. We demonstrate that RPI-GLYT2-82 dissociates from GlyT2 faster than ORG25543, providing analgesia in mouse neuropathic pain models without on-target side-effects or addiction liability. Our data provide a mechanistic understanding of allosteric inhibition of glycine transport, enabling structure-based design of non-opioid analgesics.
History
DepositionApr 26, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 25, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium- and chloride-dependent glycine transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,1104
Polymers68,9761
Non-polymers1343
Water362
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Sodium- and chloride-dependent glycine transporter 2 / GlyT-2 / GlyT2 / Solute carrier family 6 member 5


Mass: 68976.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A5, GLYT2, NET1 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q9Y345
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GLY / GLYCINE


Type: peptide linking / Mass: 75.067 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H5NO2 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: Human glycine transporter GlyT2 bound to substrate glycine
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F / Plasmid: pCDNA3.1
Buffer solutionpH: 7.5
Details: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.008 % (w/v) GDN, 0.008 % (w/v) LMNG, 0.0016 % (w/v) CHS, 1 mM glycine
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.87 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14962
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1PHENIX1.21.1_5286:model refinement
3cryoSPARCv.4.5.3particle selection
7cryoSPARCv.4.5.33D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3363288
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162255 / Symmetry type: POINT
Atomic model buildingAccession code: AF-Q9Y345-F1 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034497
ELECTRON MICROSCOPYf_angle_d0.7286131
ELECTRON MICROSCOPYf_dihedral_angle_d7.726592
ELECTRON MICROSCOPYf_chiral_restr0.046687
ELECTRON MICROSCOPYf_plane_restr0.016743

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