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- PDB-8ta2: Cryo-EM structure of the human CLC-2 chloride channel transmembra... -

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Basic information

Entry
Database: PDB / ID: 8ta2
TitleCryo-EM structure of the human CLC-2 chloride channel transmembrane domain with bound inhibitor AK-42
ComponentsChloride channel protein 2
KeywordsTRANSPORT PROTEIN/INHIBITOR / Chloride / Channel / Inhibitor / Protein / Voltage gated / TRANSPORT PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / regulation of membrane depolarization during action potential / volume-sensitive chloride channel activity / astrocyte end-foot / stabilization of membrane potential / acinar cell differentiation / cellular hypotonic response / regulation of resting membrane potential / voltage-gated chloride channel activity ...regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / regulation of membrane depolarization during action potential / volume-sensitive chloride channel activity / astrocyte end-foot / stabilization of membrane potential / acinar cell differentiation / cellular hypotonic response / regulation of resting membrane potential / voltage-gated chloride channel activity / axon initial segment / chloride channel regulator activity / dendritic spine membrane / chloride transport / phagocytosis, engulfment / positive regulation of oligodendrocyte differentiation / chloride channel complex / lung development / Stimuli-sensing channels / myelin sheath / retina development in camera-type eye / perikaryon / basolateral plasma membrane / postsynaptic membrane / plasma membrane
Similarity search - Function
Chloride channel ClC-2 / : / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily / CBS domain profile.
Similarity search - Domain/homology
Chem-GH6 / Chloride channel protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsXu, M. / Neelands, T. / Powers, A.S. / Liu, Y. / Miller, S. / Pintilie, G. / Du Bois, J. / Dror, R.O. / Chiu, W. / Maduke, M.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129541 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS113611 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS125767 United States
CitationJournal: Elife / Year: 2024
Title: CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism.
Authors: Mengyuan Xu / Torben Neelands / Alexander S Powers / Yan Liu / Steven D Miller / Grigore D Pintilie / J Du Bois / Ron O Dror / Wah Chiu / Merritt Maduke /
Abstract: CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated ...CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl-permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl-permeation pathway.
History
DepositionJun 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.0Jan 31, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Jan 31, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Jan 31, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: Database references / Category: citation / citation_author
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Revision 1.2May 28, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chloride channel protein 2
B: Chloride channel protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,6316
Polymers104,7812
Non-polymers8494
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Chloride channel protein 2 / ClC-2


Mass: 52390.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLCN2 / Production host: Homo sapiens (human) / References: UniProt: P51788
#2: Chemical ChemComp-GH6 / 2-[[2,6-bis(chloranyl)-3-phenylmethoxy-phenyl]amino]pyridine-3-carboxylic acid


Mass: 389.232 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H14Cl2N2O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
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: Chloride channel protein 2 with inhibitor AK-42 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 5.6 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 14300

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.2particle selection
2EPU2image acquisition
4cryoSPARC3.2CTF correction
7Coot0.9model fitting
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
11cryoSPARC3.2classification
12cryoSPARC3.23D reconstruction
13UCSF ChimeraX1.6model refinement
20PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5214695
3D reconstructionResolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2391813 / Num. of class averages: 10 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Q-score
Atomic model buildingAccession code: 6qvc / Source name: SwissModel / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037375
ELECTRON MICROSCOPYf_angle_d0.47510033
ELECTRON MICROSCOPYf_dihedral_angle_d6.109993
ELECTRON MICROSCOPYf_chiral_restr0.0361175
ELECTRON MICROSCOPYf_plane_restr0.0051230

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