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- EMDB-18457: Connexin-32 hemichannel upon addition of 2-aminoetoxydiphenyl borate -

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Basic information

Entry
Database: EMDB / ID: EMD-18457
TitleConnexin-32 hemichannel upon addition of 2-aminoetoxydiphenyl borate
Map data
Sample
  • Complex: Connexin-32 gap junction channel in complex with 2-aminoethoxydipheniyl borate
    • Protein or peptide: Gap junction beta-1 protein
  • Ligand: CHOLESTEROL
Keywordscell communication / channel / MEMBRANE PROTEIN
Function / homology
Function and homology information


Oligomerization of connexins into connexons / Transport of connexins along the secretory pathway / gap junction assembly / connexin complex / gap junction channel activity / Gap junction assembly / cell-cell signaling / nervous system development / endoplasmic reticulum membrane / identical protein binding
Similarity search - Function
Gap junction beta-1 protein (Cx32) / Connexin / Connexin, N-terminal / Connexin, conserved site / Gap junction protein, cysteine-rich domain / Connexin, N-terminal domain superfamily / Connexin / Connexins signature 1. / Connexins signature 2. / Connexin homologues / Gap junction channel protein cysteine-rich domain
Similarity search - Domain/homology
Gap junction beta-1 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsLavriha P / Korkhov VM / Han Y
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation184951 Switzerland
CitationJournal: PLoS One / Year: 2024
Title: Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl borate.
Authors: Pia Lavriha / Yufei Han / Xinyue Ding / Dina Schuster / Chao Qi / Anand Vaithia / Paola Picotti / Volodymyr M Korkhov /
Abstract: Gap junction intercellular communication (GJIC) between two adjacent cells involves direct exchange of cytosolic ions and small molecules via connexin gap junction channels (GJCs). Connexin GJCs have ...Gap junction intercellular communication (GJIC) between two adjacent cells involves direct exchange of cytosolic ions and small molecules via connexin gap junction channels (GJCs). Connexin GJCs have emerged as drug targets, with small molecule connexin inhibitors considered a viable therapeutic strategy in several diseases. The molecular mechanisms of GJC inhibition by known small molecule connexin inhibitors remain unknown, preventing the development of more potent and connexin-specific therapeutics. Here we show that two GJC inhibitors, mefloquine (MFQ) and 2-aminoethoxydiphenyl borate (2APB) bind to Cx32 and block dye permeation across Cx32 hemichannels (HCs) and GJCs. Cryo-EM analysis shows that 2APB binds to "site A", close to the N-terminal gating helix of Cx32 GJC, restricting the entrance to the channel pore. In contrast, MFQ binds to a distinct "site M", deeply buried within the pore. MFQ binding to this site modifies the electrostatic properties of Cx32 pore. Mutagenesis of V37, a key residue located in the site M, renders Cx32 HCs and GJCs insensitive to MFQ-mediated inhibition. Moreover, our cryo-EM analysis, mutagenesis and activity assays show that MFQ targets the M site in Cx43 GJC similarly to Cx32. Taken together, our results point to a conserved inhibitor binding site in connexin channels, opening a new route for development of specific drugs targeting connexins.
History
DepositionSep 14, 2023-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18457.png

Downloads & links

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Map

FileDownload / File: emd_18457.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 384 pix.
= 249.6 Å		0.65 Å/pix.
x 384 pix.
= 249.6 Å		0.65 Å/pix.
x 384 pix.
= 249.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.65 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.0057510836 - 0.017849414
Average (Standard dev.)0.00017353042 (±0.00080902333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18457_msk_1.map
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Half map: half1

Fileemd_18457_half_map_1.map
Annotationhalf1
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Half map: #1

Fileemd_18457_half_map_2.map
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Sample components

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Entire : Connexin-32 gap junction channel in complex with 2-aminoethoxydip...

EntireName: Connexin-32 gap junction channel in complex with 2-aminoethoxydipheniyl borate
Components
  • Complex: Connexin-32 gap junction channel in complex with 2-aminoethoxydipheniyl borate
    • Protein or peptide: Gap junction beta-1 protein
  • Ligand: CHOLESTEROL

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Supramolecule #1: Connexin-32 gap junction channel in complex with 2-aminoethoxydip...

SupramoleculeName: Connexin-32 gap junction channel in complex with 2-aminoethoxydipheniyl borate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Gap junction beta-1 protein

MacromoleculeName: Gap junction beta-1 protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.065533 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNWTGLYTLL SGVNRHSTAI GRVWLSVIFI FRIMVLVVAA ESVWGDEKSS FICNTLQPGC NSVCYDQFFP ISHVRLWSLQ LILVSTPAL LVAMHVAHQQ HIEKKMLRLE GHGDPLHLEE VKRHKVHISG TLWWTYVISV VFRLLFEAVF MYVFYLLYPG Y AMVRLVKC ...String:
MNWTGLYTLL SGVNRHSTAI GRVWLSVIFI FRIMVLVVAA ESVWGDEKSS FICNTLQPGC NSVCYDQFFP ISHVRLWSLQ LILVSTPAL LVAMHVAHQQ HIEKKMLRLE GHGDPLHLEE VKRHKVHISG TLWWTYVISV VFRLLFEAVF MYVFYLLYPG Y AMVRLVKC DVYPCPNTVD CFVSRPTEKT VFTVFMLAAS GICIILNVAE VVYLIIRACA RRAQRRSNPP SRKGSGFGHR LS PEYKQNE INKLLSEQDG SLKDILRRSP GTGAGLAEKS DRCSAC

UniProtKB: Gap junction beta-1 protein

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Macromolecule #2: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 2 / Number of copies: 6 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
25.0 mMTris-HCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / 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: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

15010

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 150125
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationSoftware - Name: RELION (ver. 4.0)

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