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- PDB-8eq4: Human PAC in nanodisc at pH 4.0 with PI(4,5)P2 diC8 -

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Basic information

Entry
Database: PDB / ID: 8eq4
TitleHuman PAC in nanodisc at pH 4.0 with PI(4,5)P2 diC8
ComponentsProton-activated chloride channel
KeywordsMEMBRANE PROTEIN / PAC / TMEM206 / ASOR / PAORAC / ion channels / chloride channel
Function / homologypH-gated chloride channel activity / TMEM206 protein / TMEM206 protein family / chloride transport / chloride channel complex / cell surface / plasma membrane / Chem-PIO / Proton-activated chloride channel
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsRuan, Z. / Lu, W.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proton-activated chloride channel
B: Proton-activated chloride channel
C: Proton-activated chloride channel
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,17018
Polymers120,2763
Non-polymers4,89415
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Proton-activated chloride channel / hPAC / Acid-sensitive outwardly-rectifying anion channel / ASOR / Proton-activated outwardly ...hPAC / Acid-sensitive outwardly-rectifying anion channel / ASOR / Proton-activated outwardly rectifying anion channel / PAORAC / Transmembrane protein 206 / hTMEM206


Mass: 40092.047 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PACC1, C1orf75, TMEM206 / Production host: Homo sapiens (human) / References: UniProt: Q9H813
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical 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 / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C25H49O19P3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Proton activated chloride channel at pH 4.0 with 0.5mM PIP2
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 4
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K

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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 FIELDBright-field microscopy / Nominal defocus max: 24000 nm / Nominal defocus min: 6000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84149 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027131
ELECTRON MICROSCOPYf_angle_d0.4619684
ELECTRON MICROSCOPYf_dihedral_angle_d9.0341110
ELECTRON MICROSCOPYf_chiral_restr0.0451113
ELECTRON MICROSCOPYf_plane_restr0.0031191

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