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- EMDB-41126: Cryo-EM structure of the human CLC-2 chloride channel transmembra... -

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Basic information

Entry
Database: EMDB / ID: EMD-41126
TitleCryo-EM structure of the human CLC-2 chloride channel transmembrane domain with bound inhibitor AK-42
Map data
Sample
  • Complex: Chloride channel protein 2 with inhibitor AK-42
    • Protein or peptide: Chloride channel protein 2
  • Ligand: 2-[[2,6-bis(chloranyl)-3-phenylmethoxy-phenyl]amino]pyridine-3-carboxylic acid
  • Ligand: CHLORIDE ION
KeywordsChloride / 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 / stabilization of membrane potential / astrocyte end-foot / 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 / stabilization of membrane potential / astrocyte end-foot / acinar cell differentiation / cellular hypotonic response / regulation of resting membrane potential / voltage-gated chloride channel activity / axon initial segment / dendritic spine membrane / chloride channel regulator activity / 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 / basolateral plasma membrane / perikaryon / 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
Chloride channel protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsXu M / Neelands T / Powers AS / Liu Y / Miller S / Pintilie G / Du Bois J / Dror RO / Chiu W / Maduke M
Funding support United States, 4 items
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, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41126.png

Downloads & links

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Map

FileDownload / File: emd_41126.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 256 pix.
= 242.176 Å		0.95 Å/pix.
x 256 pix.
= 242.176 Å		0.95 Å/pix.
x 256 pix.
= 242.176 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.946 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.2
Minimum - Maximum-4.701792 - 7.145223
Average (Standard dev.)0.0007636922 (±0.15254693)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 242.176 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41126_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41126_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Chloride channel protein 2 with inhibitor AK-42

EntireName: Chloride channel protein 2 with inhibitor AK-42
Components
  • Complex: Chloride channel protein 2 with inhibitor AK-42
    • Protein or peptide: Chloride channel protein 2
  • Ligand: 2-[[2,6-bis(chloranyl)-3-phenylmethoxy-phenyl]amino]pyridine-3-carboxylic acid
  • Ligand: CHLORIDE ION

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Supramolecule #1: Chloride channel protein 2 with inhibitor AK-42

SupramoleculeName: Chloride channel protein 2 with inhibitor AK-42 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Chloride channel protein 2

MacromoleculeName: Chloride channel protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.390672 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GEDWIFLVLL GLLMALVSWV MDYAIAACLQ AQQWMSRGLN TSILLQYLAW VTYPVVLITF SAGFTQILAP QAVGSGIPEM KTILRGVVL KEYLTLKTFI AKVIGLTCAL GSGMPLGKEG PFVHIASMCA ALLSKFLSLF GGIYENESRN TEMLAAACAV G VGCCFAAP ...String:
GEDWIFLVLL GLLMALVSWV MDYAIAACLQ AQQWMSRGLN TSILLQYLAW VTYPVVLITF SAGFTQILAP QAVGSGIPEM KTILRGVVL KEYLTLKTFI AKVIGLTCAL GSGMPLGKEG PFVHIASMCA ALLSKFLSLF GGIYENESRN TEMLAAACAV G VGCCFAAP IGGVLFSIEV TSTFFAVRNY WRGFFAATFS AFIFRVLAVW NRDEETITAL FKTRFRLDFP FDLQELPAFA VI GIASGFG GALFVYLNRK IVQVMRKQKT INRFLMRKRL LFPALVTLLI STLTFPPGFG QFMAGQLSQK ETLVTLFDNR TWV RQGLVE ELEPPSTSQA WNPPRANVFL TLVIFILMKF WMSALATTIP VPCGAFMPVF VIGAAFGRLV GESMAAWFPD GIHT DSSTY RIVPGGYAVV GAAALAGAVT HTVSTAVIVF ELTGQIAHIL PVMIAVILAN AVAQSLQPSL YDSIIRIKKL PYLP

UniProtKB: Chloride channel protein 2

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Macromolecule #2: 2-[[2,6-bis(chloranyl)-3-phenylmethoxy-phenyl]amino]pyridine-3-ca...

MacromoleculeName: 2-[[2,6-bis(chloranyl)-3-phenylmethoxy-phenyl]amino]pyridine-3-carboxylic acid
type: ligand / ID: 2 / Number of copies: 2 / Formula: GH6
Molecular weightTheoretical: 389.232 Da
Chemical component information

ChemComp-GH6:
2-[[2,6-bis(chloranyl)-3-phenylmethoxy-phenyl]amino]pyridine-3-carboxylic acid

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Macromolecule #3: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 14300 / Average exposure time: 5.6 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5214695
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 10 / Resolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 2391813
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC (ver. 3.2)

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Atomic model buiding 1

Initial modelPDB ID:

6qvc


Chain - Source name: SwissModel / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Q-score
Output model

PDB-8ta2:
Cryo-EM structure of the human CLC-2 chloride channel transmembrane domain with bound inhibitor AK-42

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