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- EMDB-28535: Human PAC in nanodisc at pH 4.0 with PI(4,5)P2 diC8 -

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Basic information

Entry
Database: EMDB / ID: EMD-28535
TitleHuman PAC in nanodisc at pH 4.0 with PI(4,5)P2 diC8
Map dataUnsharpened map of human PAC in nanodisc at pH4 with 0.5mM PI(4,5)P2 diC8.
Sample
  • Complex: Proton activated chloride channel at pH 4.0 with 0.5mM PIP2
    • Protein or peptide: Proton-activated chloride channel
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
Function / homologypH-gated chloride channel activity / TMEM206 protein / TMEM206 protein family / chloride transport / chloride channel complex / cell surface / plasma membrane / Proton-activated chloride channel
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsRuan Z / Lu W
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)K99NS128258 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01NS112363 United States
American Heart Association20POST35120556 United States
CitationJournal: Elife / Year: 2023
Title: Inhibition of the proton-activated chloride channel PAC by PIP.
Authors: Ljubica Mihaljević / Zheng Ruan / James Osei-Owusu / Wei Lü / Zhaozhu Qiu /
Abstract: Proton-activated chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel, encoded by (). PAC regulates endosomal acidification and macropinosome shrinkage by releasing chloride ...Proton-activated chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel, encoded by (). PAC regulates endosomal acidification and macropinosome shrinkage by releasing chloride from the organelle lumens. It is also found at the cell surface, where it is activated under pathological conditions related to acidosis and contributes to acid-induced cell death. However, the pharmacology of the PAC channel is poorly understood. Here, we report that phosphatidylinositol (4,5)-bisphosphate (PIP) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP at pH 4.0 and identified its putative binding site, which, surprisingly, locates on the extracellular side of the transmembrane domain (TMD). While the overall conformation resembles the previously resolved PAC structure in the desensitized state, the TMD undergoes remodeling upon PIP-binding. Structural and electrophysiological analyses suggest that PIP inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. Our findings identify PIP as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel.
History
DepositionOct 7, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28535.png

Downloads & links

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Map

FileDownload / File: emd_28535.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map of human PAC in nanodisc at pH4 with 0.5mM PI(4,5)P2 diC8.
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.111
Minimum - Maximum-0.32024676 - 0.80025566
Average (Standard dev.)0.0031507025 (±0.021867597)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.79999 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map of human PAC in nanodisc at...

Fileemd_28535_additional_1.map
AnnotationSharpened map of human PAC in nanodisc at pH4 with 0.5mM PI(4,5)P2 diC8.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_28535_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28535_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Proton activated chloride channel at pH 4.0 with 0.5mM PIP2

EntireName: Proton activated chloride channel at pH 4.0 with 0.5mM PIP2
Components
  • Complex: Proton activated chloride channel at pH 4.0 with 0.5mM PIP2
    • Protein or peptide: Proton-activated chloride channel
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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Supramolecule #1: Proton activated chloride channel at pH 4.0 with 0.5mM PIP2

SupramoleculeName: Proton activated chloride channel at pH 4.0 with 0.5mM PIP2
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Proton-activated chloride channel

MacromoleculeName: Proton-activated chloride channel / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.092047 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MIRQERSTSY QELSEELVQV VENSELADEQ DKETVRVQGP GILPGLDSES ASSSIRFSKA CLKNVFSVLL IFIYLLLMAV AVFLVYRTI TDFREKLKHP VMSVSYKEVD RYDAPGIALY PGQAQLLSCK HHYEVIPPLT SPGQPGDMNC TTQRINYTDP F SNQTVKSA ...String:
MIRQERSTSY QELSEELVQV VENSELADEQ DKETVRVQGP GILPGLDSES ASSSIRFSKA CLKNVFSVLL IFIYLLLMAV AVFLVYRTI TDFREKLKHP VMSVSYKEVD RYDAPGIALY PGQAQLLSCK HHYEVIPPLT SPGQPGDMNC TTQRINYTDP F SNQTVKSA LIVQGPREVK KRELVFLQFR LNKSSEDFSA IDYLLFSSFQ EFLQSPNRVG FMQACESAYS SWKFSGGFRT WV KMSLVKT KEEDGREAVE FRQETSVVNY IDQRPAAKKS AQLFFVVFEW KDPFIQKVQD IVTANPWNTI ALLCGAFLAL FKA AEFAKL SIKWMIKIRK RYLKRRGQAT SHIS

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 12 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #3: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

MacromoleculeName: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
type: ligand / ID: 3 / Number of copies: 3 / Formula: PIO
Molecular weightTheoretical: 746.566 Da
Chemical component information

ChemComp-PIO:
[(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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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: 4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 24.0 µm / Nominal defocus min: 6.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 84149

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