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- PDB-6wth: Full-length human ENaC ECD -

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Basic information

Entry
Database: PDB / ID: 6wth
TitleFull-length human ENaC ECD
Components
  • (Amiloride-sensitive sodium channel subunit ...) x 3
  • 10D4 Fab
  • 7B1 Fab
KeywordsMEMBRANE PROTEIN / sodium channel / blood pressure / epithelial / salt transport
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 / sperm principal piece / cellular response to aldosterone / sodium channel complex ...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 / sperm principal piece / 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 / motile cilium / response to food / erythrocyte homeostasis / ciliary membrane / WW domain binding / monoatomic ion channel activity / acrosomal vesicle / sodium ion transmembrane transport / cytoplasmic vesicle membrane / Stimuli-sensing channels / regulation of blood pressure / multicellular organism growth / gene expression / apical plasma membrane / response to xenobiotic stimulus / external side of plasma membrane / extracellular exosome / nucleoplasm / plasma membrane / cytosol / cytoplasm
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 alpha / Epithelial sodium channel subunit beta / Epithelial sodium channel subunit gamma
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsPosert, R. / Baconguis, I. / Noreng, S. / Bharadwaj, A. / Houser, A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/Office of the DirectorDP5OD017871 United States
American Heart Association19TPA34760754 United States
American Heart Association18PRE33990205 United States
National Science Foundation (NSF, United States)DGE-1937961 United States
CitationJournal: Elife / Year: 2020
Title: Molecular principles of assembly, activation, and inhibition in epithelial sodium channel.
Authors: Sigrid Noreng / Richard Posert / Arpita Bharadwaj / Alexandra Houser / Isabelle Baconguis /
Abstract: The molecular bases of heteromeric assembly and link between Na self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated ...The molecular bases of heteromeric assembly and link between Na self-inhibition and protease-sensitivity in epithelial sodium channels (ENaCs) are not fully understood. Previously, we demonstrated that ENaC subunits - α, β, and γ - assemble in a counterclockwise configuration when viewed from outside the cell with the protease-sensitive GRIP domains in the periphery (Noreng et al., 2018). Here we describe the structure of ENaC resolved by cryo-electron microscopy at 3 Å. We find that a combination of precise domain arrangement and complementary hydrogen bonding network defines the subunit arrangement. Furthermore, we determined that the α subunit has a primary functional module consisting of the finger and GRIP domains. The module is bifurcated by the α2 helix dividing two distinct regulatory sites: Na and the inhibitory peptide. Removal of the inhibitory peptide perturbs the Na site via the α2 helix highlighting the critical role of the α2 helix in regulating ENaC function.
History
DepositionMay 2, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Amiloride-sensitive sodium channel subunit alpha
B: Amiloride-sensitive sodium channel subunit beta
C: Amiloride-sensitive sodium channel subunit gamma
D: 7B1 Fab
E: 7B1 Fab
F: 10D4 Fab
G: 10D4 Fab
hetero molecules


Theoretical massNumber of molelcules
Total (without water)264,77415
Polymers262,5937
Non-polymers2,1818
Water00
1


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

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Components

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Amiloride-sensitive sodium channel subunit ... , 3 types, 3 molecules ABC

#1: Protein Amiloride-sensitive sodium channel subunit alpha / Alpha-NaCH / Epithelial Na(+) channel subunit alpha / ENaCA / Nonvoltage-gated sodium channel 1 ...Alpha-NaCH / Epithelial Na(+) channel subunit alpha / ENaCA / Nonvoltage-gated sodium channel 1 subunit alpha / SCNEA


Mass: 75780.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1A, SCNN1 / Production host: Homo sapiens (human) / References: UniProt: P37088
#2: Protein Amiloride-sensitive sodium channel subunit beta / Beta-NaCH / Epithelial Na(+) channel subunit beta / ENaCB / Nonvoltage-gated sodium channel 1 ...Beta-NaCH / Epithelial Na(+) channel subunit beta / ENaCB / Nonvoltage-gated sodium channel 1 subunit beta / SCNEB


Mass: 72728.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1B / Production host: Homo sapiens (human) / References: UniProt: P51168
#3: Protein Amiloride-sensitive sodium channel subunit gamma / Epithelial Na(+) channel subunit gamma / Gamma-ENaC / Gamma-NaCH / Nonvoltage-gated sodium channel ...Epithelial Na(+) channel subunit gamma / Gamma-ENaC / Gamma-NaCH / Nonvoltage-gated sodium channel 1 subunit gamma / SCNEG


Mass: 74352.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SCNN1G / Production host: Homo sapiens (human) / References: UniProt: P51170

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Antibody , 2 types, 4 molecules DEFG

#4: Antibody 7B1 Fab


Mass: 10060.393 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#5: Antibody 10D4 Fab


Mass: 9805.078 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Sugars , 2 types, 7 molecules

#6: 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.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#8: 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.: 4
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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Non-polymers , 1 types, 1 molecules

#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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

ComponentName: Full-length human ENaC heterotrimer bound with two high-affinity Fabs
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.32 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293T/17 / Plasmid: pBacMam
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285 K
Details: 3.5 uL applied, manual blot, fresh 3.5 uL applied, vitrobot blot and freeze

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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 FIELD
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.1_3865: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 252071 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211972
ELECTRON MICROSCOPYf_angle_d0.3916309
ELECTRON MICROSCOPYf_dihedral_angle_d12.2744046
ELECTRON MICROSCOPYf_chiral_restr0.0461884
ELECTRON MICROSCOPYf_plane_restr0.0022128

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