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- PDB-9bku: Cryo-EM structure of TRPV3 K169A in nanodiscs incubated with NASPM -

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Basic information

Entry
Database: PDB / ID: 9bku
TitleCryo-EM structure of TRPV3 K169A in nanodiscs incubated with NASPM
ComponentsTransient receptor potential cation channel subfamily V member 3
KeywordsMEMBRANE PROTEIN / tetramer / transient receptor
Function / homology
Function and homology information


negative regulation of hair cycle / osmosensory signaling pathway / TRP channels / response to temperature stimulus / sodium channel activity / positive regulation of calcium ion import / calcium ion import across plasma membrane / actin filament organization / calcium ion transmembrane transport / calcium channel activity ...negative regulation of hair cycle / osmosensory signaling pathway / TRP channels / response to temperature stimulus / sodium channel activity / positive regulation of calcium ion import / calcium ion import across plasma membrane / actin filament organization / calcium ion transmembrane transport / calcium channel activity / lysosome / receptor complex / cilium / metal ion binding / identical protein binding / plasma membrane / cytoplasm
Similarity search - Function
Transient receptor potential cation channel subfamily V member 1-4 / Transient receptor potential cation channel subfamily V / Ankyrin repeat / 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
Chem-6OU / Transient receptor potential cation channel subfamily V member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsZhang, J. / Yuan, P. / Maksaev, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R35HL171542 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS099341 United States
CitationJournal: Commun Biol / Year: 2025
Title: Molecular basis of TRPV3 channel blockade by intracellular polyamines.
Authors: Jingying Zhang / Peng Yuan / Colin G Nichols / Grigory Maksaev /
Abstract: ThermoTRPV1-4 channels are involved in the regulation of multiple physiological processes, including thermo- and pain perception, thermoregulation, itch, and nociception and therefore tight control ...ThermoTRPV1-4 channels are involved in the regulation of multiple physiological processes, including thermo- and pain perception, thermoregulation, itch, and nociception and therefore tight control of their activity is a critical requirement for correct perception of noxious stimuli and pain. We previously reported a voltage-dependent inhibition of TRPV1-4 channels by intracellular polyamines that could be explained by high affinity spermine binding in, and passage through, the permeation path. Here, using electrophysiology and cryo-electron microscopy, we elucidate molecular details of TRPV3 blockade by endogenous spermine and its analog NASPM. We identify a high-affinity polyamine interaction site at the intracellular side of the pore, formed by residues E679 and E682, with no significant contribution of residues at the channel selectivity filter. A cryo-EM structure of TRPV3 in the presence of NASPM reveals conformational changes coupled to polyamine blockade. Paradoxically, although the TRPV3 'gating switch' is in the 'activated' configuration, the pore is closed at both gates. A modified blocking model, in which spermine interacts with the cytoplasmic entrance to the channel, from which spermine may permeate, or cause closure of the channel, provides a unifying explanation for electrophysiological and structural data and furnishes the essential background for further exploitation of this regulatory process.
History
DepositionApr 29, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 21, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily V member 3
B: Transient receptor potential cation channel subfamily V member 3
C: Transient receptor potential cation channel subfamily V member 3
D: Transient receptor potential cation channel subfamily V member 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)392,60340
Polymers366,7554
Non-polymers25,84836
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "D"
d_4ens_1chain "C"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ARGARGPHEPHEAA117 - 748117 - 748
d_126OU6OU6OU6OUAE801
d_136OU6OU6OU6OUBN801
d_146OU6OU6OU6OUAF802
d_156OU6OU6OU6OUDFA801
d_166OU6OU6OU6OUAG803
d_176OU6OU6OU6OUAH804
d_186OU6OU6OU6OUAI805
d_196OU6OU6OU6OUAJ806
d_1106OU6OU6OU6OUBO802
d_21ARGARGPHEPHEBB117 - 748117 - 748
d_226OU6OU6OU6OUBP803
d_236OU6OU6OU6OUCW801
d_246OU6OU6OU6OUBQ804
d_256OU6OU6OU6OUAK807
d_266OU6OU6OU6OUBR805
d_276OU6OU6OU6OUBS806
d_286OU6OU6OU6OUBT807
d_296OU6OU6OU6OUBU808
d_2106OU6OU6OU6OUCX802
d_31ARGARGPHEPHEDD117 - 748117 - 748
d_326OU6OU6OU6OUDIA804
d_336OU6OU6OU6OUAL808
d_346OU6OU6OU6OUDJA805
d_356OU6OU6OU6OUCEA809
d_366OU6OU6OU6OUDKA806
d_376OU6OU6OU6OUDLA807
d_386OU6OU6OU6OUDMA808
d_396OU6OU6OU6OUDNA809
d_3106OU6OU6OU6OUAM809
d_41ARGARGPHEPHECC117 - 748117 - 748
d_426OU6OU6OU6OUCY803
d_436OU6OU6OU6OUDGA802
d_446OU6OU6OU6OUCZ804
d_456OU6OU6OU6OUBV809
d_466OU6OU6OU6OUCAA805
d_476OU6OU6OU6OUCBA806
d_486OU6OU6OU6OUCCA807
d_496OU6OU6OU6OUCDA808
d_4106OU6OU6OU6OUDHA803

NCS oper:
IDCodeMatrixVector
1given(6.35047570086E-14, -1, -1.79830507304E-13), (1, 6.35047570086E-14, -5.0024304701E-13), (5.0024304701E-13, -1.79830507304E-13, 1)336, 7.59143858886E-11, -6.86668499839E-11
2given(-2.32858177185E-12, 1, 6.21182115952E-12), (-1, -2.32858177185E-12, 1.75520790686E-11), (1.75520790686E-11, -6.21182115948E-12, 1)-6.19820639258E-10, 335.999999997, -2.43176145887E-9
3given(-1, -2.34922099399E-12, 1.76771391952E-11), (2.34922099387E-12, -1, -6.25367082094E-12), (1.76771391952E-11, -6.2536708209E-12, 1)335.999999997, 336.000000001, -2.44938291871E-9

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Components

#1: Protein
Transient receptor potential cation channel subfamily V member 3 / TrpV3 / Vanilloid receptor-like 3 / VRL-3


Mass: 91688.805 Da / Num. of mol.: 4 / Mutation: K169A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPV3 / Production host: Komagataella pastoris (fungus) / References: UniProt: Q8NET8
#2: Chemical...
ChemComp-6OU / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate


Mass: 717.996 Da / Num. of mol.: 36 / Source method: obtained synthetically / Formula: C39H76NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY
Has protein modificationN
Sequence detailsThe sequence for this construct was obtained from GenBank sequence EAW90503.1.

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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: TRPV3 K169A in nanodiscs incubated with NASPM / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl PH 8.0, 150 mM NaCl, 0.1 mM NASPM
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMTris1
30.1 mM1-Naphthylacetyl spermine trihydrochloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 44.1 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 3749

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Processing

EM software
IDNameVersionCategory
12Coot0.9model fitting
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1864667
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 266529 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6UW6

6uw6


Accession code: 6UW6 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 60.38 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001817804
ELECTRON MICROSCOPYf_angle_d0.395724128
ELECTRON MICROSCOPYf_chiral_restr0.03232876
ELECTRON MICROSCOPYf_plane_restr0.00272976
ELECTRON MICROSCOPYf_dihedral_angle_d10.07182922
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAELECTRON MICROSCOPYNCS constraints2.68062868236E-10
ens_1d_3AAELECTRON MICROSCOPYNCS constraints2.68060047771E-10
ens_1d_4AAELECTRON MICROSCOPYNCS constraints7.36312732167E-13

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