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- PDB-8f79: LRRC8A(T48D):C conformation 2 top focus -

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Basic information

Entry
Database: PDB / ID: 8f79
TitleLRRC8A(T48D):C conformation 2 top focus
Components
  • Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562
  • Volume-regulated anion channel subunit LRRC8C
KeywordsMEMBRANE PROTEIN / ion channel / volume-regulation
Function / homology
Function and homology information


Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / aspartate transmembrane transport / taurine transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transmembrane transport / cellular response to osmotic stress / protein hexamerization ...Miscellaneous transport and binding events / pre-B cell differentiation / volume-sensitive anion channel activity / aspartate transmembrane transport / taurine transmembrane transport / cyclic-GMP-AMP transmembrane transporter activity / cyclic-GMP-AMP transmembrane import across plasma membrane / monoatomic anion transmembrane transport / cellular response to osmotic stress / protein hexamerization / cell volume homeostasis / monoatomic anion transport / response to osmotic stress / monoatomic ion channel complex / fat cell differentiation / intracellular glucose homeostasis / positive regulation of myoblast differentiation / chloride transmembrane transport / electron transport chain / positive regulation of insulin secretion / spermatogenesis / electron transfer activity / periplasmic space / iron ion binding / lysosomal membrane / heme binding / endoplasmic reticulum membrane / cell surface / endoplasmic reticulum / identical protein binding / plasma membrane
Similarity search - Function
LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / : / Leucine Rich Repeat / Leucine-rich repeat, SDS22-like subfamily / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Leucine rich repeat / Leucine-rich repeat, typical subtype ...LRRC8, pannexin-like TM region / Pannexin-like TM region of LRRC8 / : / Leucine Rich Repeat / Leucine-rich repeat, SDS22-like subfamily / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Soluble cytochrome b562 / Volume-regulated anion channel subunit LRRC8A / Volume-regulated anion channel subunit LRRC8C
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsKern, D.M. / Brohawn, S.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM128263 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels.
Authors: David M Kern / Julia Bleier / Somnath Mukherjee / Jennifer M Hill / Anthony A Kossiakoff / Ehud Y Isacoff / Stephen G Brohawn /
Abstract: Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume-regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate ...Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume-regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (SWELL1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents across cell types. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here we leverage a fiducial-tagging strategy to determine single-particle cryo-EM structures of heterohexameric LRRC8A:C channels in multiple conformations. Compared to homomers, LRRC8A:C channels show pronounced differences in architecture due to heterotypic LRR interactions that displace subunits away from the conduction axis and poise the channel for activation. Structures and functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. These results provide insight into determinants for heteromeric LRRC8 channel assembly, activity and gating by lipids.
History
DepositionNov 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 29, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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 LRRC8A,Soluble cytochrome b562
B: Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562
C: Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562
D: Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562
E: Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562
F: Volume-regulated anion channel subunit LRRC8C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)633,99423
Polymers621,3456
Non-polymers12,64917
Water1086
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Volume-regulated anion channel subunit LRRC8A,Soluble cytochrome b562 / Leucine-rich repeat-containing protein 8A / Protein ebouriffe / ebo / Cytochrome b-562


Mass: 105544.086 Da / Num. of mol.: 5 / Mutation: T48D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc8a, Lrrc8, cybC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q80WG5, UniProt: P0ABE7
#2: Protein Volume-regulated anion channel subunit LRRC8C / Factor for adipocyte differentiation 158 / Leucine-rich repeat-containing protein 8C


Mass: 93624.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Lrrc8c, Ad158, Fad158 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8R502
#3: Chemical
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 17 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: LRRC8A:C / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.620 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71566 / Symmetry type: POINT
RefinementCross valid method: NONE

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