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- PDB-8dxr: Structure of LRRC8C-LRRC8A(IL125) Chimera, Class 5 -

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Basic information

Entry
Database: PDB / ID: 8dxr
TitleStructure of LRRC8C-LRRC8A(IL125) Chimera, Class 5
ComponentsVolume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A
KeywordsTRANSPORT PROTEIN / LRRC8C / LRRC8A / SWELL / VRAC / Chimera
Function / homology
Function and homology information


Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / cellular response to osmotic stress / monoatomic anion transport ...Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / taurine transmembrane transport / monoatomic anion transmembrane transport / aspartate transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / cellular response to osmotic stress / monoatomic anion transport / cell volume homeostasis / protein hexamerization / response to osmotic stress / monoatomic ion channel complex / fat cell differentiation / intracellular glucose homeostasis / positive regulation of myoblast differentiation / chloride transmembrane transport / positive regulation of insulin secretion / spermatogenesis / lysosomal membrane / endoplasmic reticulum membrane / cell surface / signal transduction / membrane / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / Leucine-rich repeat, SDS22-like subfamily / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Volume-regulated anion channel subunit LRRC8A / Volume-regulated anion channel subunit LRRC8C
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsTakahashi, H. / Yamada, T. / Denton, J.S. / Strange, K. / Karakas, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK51610 United States
CitationJournal: Elife / Year: 2023
Title: Cryo-EM structures of an LRRC8 chimera with native functional properties reveal heptameric assembly.
Authors: Hirohide Takahashi / Toshiki Yamada / Jerod S Denton / Kevin Strange / Erkan Karakas /
Abstract: Volume-regulated anion channels (VRACs) mediate volume regulatory Cl and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown ...Volume-regulated anion channels (VRACs) mediate volume regulatory Cl and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL1) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL1) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL1) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.
History
DepositionAug 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A
B: Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A
C: Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A
D: Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A
E: Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A
F: Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A
G: Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A


Theoretical massNumber of molelcules
Total (without water)663,2347
Polymers663,2347
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, SEC and native-PAGE
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Volume-regulated anion channel subunit LRRC8C,Volume-regulated anion channel subunit LRRC8A / / Factor for adipocyte differentiation 158 / Leucine-rich repeat-containing protein 8C / Leucine-rich ...Factor for adipocyte differentiation 158 / Leucine-rich repeat-containing protein 8C / Leucine-rich repeat-containing protein 8A / HsLRRC8A / Swelling protein 1


Mass: 94747.695 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: LRRC8C, AD158, FAD158, LRRC8A, KIAA1437, LRRC8, SWELL1, UNQ221/PRO247
Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8TDW0, UniProt: Q8IWT6

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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: LRRC8C-LRRC8A(IL125) chimera / Type: COMPLEX / Details: Heptameric LRRC8C-LRRC8A(IL125) chimera. / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.7 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
10.15 MSodium chlorideNaClSodium chloride1
20.05 MTris buffer pH 8.0Tris-HClTris1
30.005 %LMNG detergentLMNG1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 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: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 3198

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 846122
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85591 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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