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- PDB-6cv9: Cytoplasmic domain of mTRPC6 -

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Basic information

Entry
Database: PDB / ID: 6cv9
TitleCytoplasmic domain of mTRPC6
ComponentsShort transient receptor potential channel 6
KeywordsTRANSPORT PROTEIN / Ion Channel / Membrane Protein / TRP Channel / TRPC6 / focal segmental glomerulosclerosis
Function / homology
Function and homology information


Elevation of cytosolic Ca2+ levels / Effects of PIP2 hydrolysis / positive regulation of ion transmembrane transporter activity / manganese ion transport / slit diaphragm / negative regulation of dendrite morphogenesis / : / store-operated calcium channel activity / cation channel complex / TRP channels ...Elevation of cytosolic Ca2+ levels / Effects of PIP2 hydrolysis / positive regulation of ion transmembrane transporter activity / manganese ion transport / slit diaphragm / negative regulation of dendrite morphogenesis / : / store-operated calcium channel activity / cation channel complex / TRP channels / inositol 1,4,5 trisphosphate binding / positive regulation of calcium ion transport / actinin binding / clathrin binding / single fertilization / plasma membrane => GO:0005886 / monoatomic cation channel activity / regulation of cytosolic calcium ion concentration / positive regulation of neuron differentiation / positive regulation of peptidyl-threonine phosphorylation / calcium ion transmembrane transport / calcium channel activity / neuron differentiation / cellular response to hydrogen peroxide / monoatomic ion channel activity / actin binding / ATPase binding / positive regulation of cytosolic calcium ion concentration / cellular response to hypoxia / protein homodimerization activity / membrane / plasma membrane / cytoplasm
Similarity search - Function
Transient receptor potential channel, canonical 6 / Transient receptor ion channel domain / Transient receptor ion channel II / Domain of unknown function DUF3447 / Transient receptor potential channel, canonical / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat ...Transient receptor potential channel, canonical 6 / Transient receptor ion channel domain / Transient receptor ion channel II / Domain of unknown function DUF3447 / Transient receptor potential channel, canonical / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Short transient receptor potential channel 6
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsAzumaya, C.M. / Sierra-Valdez, F.J. / Cordero-Morales, J.F. / Nakagawa, T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD)R01HD061543 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM125629 United States
CitationJournal: J Biol Chem / Year: 2018
Title: Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).
Authors: Caleigh M Azumaya / Francisco Sierra-Valdez / Julio F Cordero-Morales / Terunaga Nakagawa /
Abstract: The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration ...The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte, and mutations in its cytoplasmic domain cause FSGS in humans. evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here, we report a cryo-EM structure of the cytoplasmic domain of murine TRPC6 at 3.8 Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family.
History
DepositionMar 27, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 23, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 11, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Short transient receptor potential channel 6
B: Short transient receptor potential channel 6
C: Short transient receptor potential channel 6
D: Short transient receptor potential channel 6


Theoretical massNumber of molelcules
Total (without water)386,6864
Polymers386,6864
Non-polymers00
Water0
1


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

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Components

#1: Protein
Short transient receptor potential channel 6 / TrpC6 / Calcium entry channel / Transient receptor protein 6 / TRP-6


Mass: 96671.375 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Trpc6, Trp6, Trrp6 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q61143

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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: cytoplasmic domain of TRPC6 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.144 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
24 mMtris(2-carboxyethyl)phosphineTCEP1
320 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse
Specimen supportDetails: 25 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 K
Details: blotted for 8 seconds, 3 filter papers on each side

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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 magnification: 89000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 0.001 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 46.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2618
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Spherical aberration corrector: Microscope was modified with a Cs aberration corrector
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.0.4CTF correction
7Coot0.8.9model fitting
9PHENIX1.13-2998-000model refinement
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 663034
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67280 / Symmetry type: POINT
Atomic model buildingB value: 113 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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