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- PDB-9blr: Human SCNN1D-SCNN1B-SCNN1G ENaC trimer -

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Basic information

Entry
Database: PDB / ID: 9blr
TitleHuman SCNN1D-SCNN1B-SCNN1G ENaC trimer
Components
  • (Amiloride-sensitive sodium channel subunit ...) x 2
  • Isoform 1 of Amiloride-sensitive sodium channel subunit delta
KeywordsMEMBRANE PROTEIN / Ion channel
Function / homology
Function and homology information


sensory perception of salty taste / Sensory perception of salty taste / sensory perception of sour taste / neutrophil-mediated killing of bacterium / aldosterone metabolic process / leukocyte activation involved in inflammatory response / cellular response to vasopressin / cellular response to aldosterone / sodium channel complex / epithelial fluid transport ...sensory perception of salty taste / Sensory perception of salty taste / sensory perception of sour taste / neutrophil-mediated killing of bacterium / aldosterone metabolic process / leukocyte activation involved in inflammatory response / cellular response to vasopressin / cellular response to aldosterone / sodium channel complex / epithelial fluid transport / mucus secretion / sodium ion homeostasis / renal system process / artery smooth muscle contraction / neutrophil activation involved in immune response / multicellular organismal-level water homeostasis / potassium ion homeostasis / intracellular sodium ion homeostasis / cellular response to acidic pH / sodium ion import across plasma membrane / ligand-gated sodium channel activity / erythrocyte homeostasis / response to food / WW domain binding / sodium channel activity / monoatomic ion channel activity / sodium ion transmembrane transport / cytoplasmic vesicle membrane / Stimuli-sensing channels / regulation of blood pressure / multicellular organism growth / actin cytoskeleton / gene expression / apical plasma membrane / response to xenobiotic stimulus / external side of plasma membrane / extracellular exosome / nucleoplasm / membrane / plasma membrane
Similarity search - Function
Epithelial sodium channel, chordates / Epithelial sodium channel, conserved site / Amiloride-sensitive sodium channels signature. / Epithelial sodium channel / Amiloride-sensitive sodium channel
Similarity search - Domain/homology
Epithelial sodium channel subunit beta / Epithelial sodium channel subunit gamma / Epithelial sodium channel subunit delta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsHouser, A. / Baconguis, I.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM138862 United States
Cystic Fibrosis FoundationBACONG22G0 United States
National Science Foundation (NSF, United States)GVPRS0015B4 United States
CitationJournal: Structure / Year: 2025
Title: Structural insights into subunit-dependent functional regulation in epithelial sodium channels.
Authors: Alexandra Houser / Isabelle Baconguis /
Abstract: Epithelial sodium channels (ENaCs) play a crucial role in Na reabsorption in mammals. To date, four subunits have been identified-α, β, γ, and δ-believed to form different heteromeric complexes. ...Epithelial sodium channels (ENaCs) play a crucial role in Na reabsorption in mammals. To date, four subunits have been identified-α, β, γ, and δ-believed to form different heteromeric complexes. Currently, only the structure of the αβγ complex is known. To investigate the formation of channels with different subunit compositions and to determine how each subunit contributes to distinct channel properties, we co-expressed human δ, β, and γ. Using single-particle cryoelectron microscopy, we observed three distinct ENaC complexes. The structures unveil a pattern in which β and γ positions are conserved among the different complexes while the α position in αβγ trimer is occupied by either δ or another β. The δ subunit induces structural rearrangements in the γ subunit, which may contribute to the differences in channel activity between αβγ and δβγ channels. These structural changes provide molecular insights into how ENaC subunit composition modulates channel function.
History
DepositionMay 1, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform 1 of Amiloride-sensitive sodium channel subunit delta
B: Amiloride-sensitive sodium channel subunit beta
C: Amiloride-sensitive sodium channel subunit gamma
hetero molecules


Theoretical massNumber of molelcules
Total (without water)220,68513
Polymers217,1733
Non-polymers3,51110
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Isoform 1 of Amiloride-sensitive sodium channel subunit delta / Delta-NaCH / Epithelial Na(+) channel subunit delta / Delta-ENaC / ENaCD / Nonvoltage-gated sodium ...Delta-NaCH / Epithelial Na(+) channel subunit delta / Delta-ENaC / ENaCD / Nonvoltage-gated sodium channel 1 subunit delta / SCNED


Mass: 70273.773 Da / Num. of mol.: 1 / Mutation: C64A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1D, DNACH / Plasmid: pGEM BACMID / Cell line (production host): HEK293S GNTI- / Production host: Homo sapiens (human) / References: UniProt: P51172

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Amiloride-sensitive sodium channel subunit ... , 2 types, 2 molecules BC

#2: Protein Amiloride-sensitive sodium channel subunit beta / Beta-NaCH / Epithelial Na(+) channel subunit beta / Beta-ENaC / ENaCB / Nonvoltage-gated sodium ...Beta-NaCH / Epithelial Na(+) channel subunit beta / Beta-ENaC / ENaCB / Nonvoltage-gated sodium channel 1 subunit beta / SCNEB


Mass: 72696.828 Da / Num. of mol.: 1 / Mutation: C30A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1B / Plasmid: pGEM BACMID / Cell line (production host): HEK293S GNTI- / Production host: Homo sapiens (human) / References: UniProt: P51168
#3: Protein Amiloride-sensitive sodium channel subunit gamma / Epithelial Na(+) channel subunit gamma / ENaCG / Gamma-ENaC / Gamma-NaCH / Nonvoltage-gated sodium ...Epithelial Na(+) channel subunit gamma / ENaCG / Gamma-ENaC / Gamma-NaCH / Nonvoltage-gated sodium channel 1 subunit gamma / SCNEG


Mass: 74202.742 Da / Num. of mol.: 1 / Mutation: C33A, C41A, R138A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1G / Plasmid: pGEM BACMID / Cell line (production host): HEK293S GNTI- / Production host: Homo sapiens (human) / References: UniProt: P51170

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Sugars , 3 types, 10 molecules

#4: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
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

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Details

Has ligand of interestN
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Amiloride-sensitive sodium channel subunits DELTA, BETA, and GAMMACELL#10NATURAL
2Human delta subunit isoform 1COMPLEX#11RECOMBINANTContains mutation C64A in the predicted transmembrane domain. C64 is thought to be palmitoylated and effect gating.
3Human beta subunit isoform 1COMPLEX#11RECOMBINANTContains mutation C30A in the predicted transmembrane domain. C30 is thought to be palmitoylated and effect gating.
4Human gamma subunit isoform 1COMPLEX#21RECOMBINANTContains mutations C33A and C41A in the predicted transmembrane domain. C333 and C41 are thought to be palmitoylated and effect gating. Additionally R138 is mutated to alanine. This residue is hypothesized to be key in gating by proteolysis.
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11
2270.2 kDa/nmNO
3372.6 kDa/nmNO
4474.1 kDa/nmNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Homo sapiens (human)9606HEK293S GNTI-
23Homo sapiens (human)9606HEK293S GNTI-
34Homo sapiens (human)9606HEK293S GNTI-
Buffer solutionpH: 7.4
Details: Vitrobot blot parameters were set to a wait time of 0s, blot time of 2s, and a blot force of 1
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMpotassium chlorideKCl1
220 mMHEPESC8H18N2O4S1
35 mMcalcium chlorideCaCl21
SpecimenConc.: 3.44 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse sample of delta, beta, and gamma ENaC with 10D4 Fab for the beta subunit
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 295.15 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: SPOT SCAN
Electron lensMode: DIFFRACTION / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18108

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.3.1particle selectionThis is the version at the end of processing
2SerialEMunkimage acquisitionmultishot multihole was used
4cryoSPARC4.3.1CTF correction
7PHENIX1.2model fitting
9cryoSPARC4.3.1initial Euler assignment
10cryoSPARC4.3.1final Euler assignment
11cryoSPARC4.3.1classification
12cryoSPARC4.3.13D reconstruction
13ISOLDE1.6model refinement
14PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1121564
Details: From blob picking in cryoSPARC with a box size of 440 with a maximum particle diameter of 165 and a minimum of 140. Minimum separation distance was 0.6 diameters
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193373 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model buildingPDB-ID: 6WTH

6wth


Accession code: 6WTH / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049716
ELECTRON MICROSCOPYf_angle_d0.72413187
ELECTRON MICROSCOPYf_dihedral_angle_d12.6643559
ELECTRON MICROSCOPYf_chiral_restr0.0491475
ELECTRON MICROSCOPYf_plane_restr0.0061676

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