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- PDB-6v1y: Cryo-EM Structure of the Hyperpolarization-Activated Potassium Ch... -

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Basic information

Entry
Database: PDB / ID: 6v1y
TitleCryo-EM Structure of the Hyperpolarization-Activated Potassium Channel KAT1: Octamer
ComponentsPotassium channel KAT1
KeywordsTRANSPORT PROTEIN / membrane protein / voltage-gated ion channel / potassium channel
Function / homology
Function and homology information


inward rectifier potassium channel activity / monoatomic ion channel complex / identical protein binding / plasma membrane
Similarity search - Function
Potassium channel KAT/AKT / KHA domain / KHA, dimerisation domain of potassium ion channel / KHA domain profile. / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily ...Potassium channel KAT/AKT / KHA domain / KHA, dimerisation domain of potassium ion channel / KHA domain profile. / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
(3beta,5beta,14beta,17alpha)-cholestan-3-ol / Chem-QNP / Potassium channel KAT1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsClark, M.D. / Contreras, G.F. / Shen, R. / Perozo, E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM088406 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)F30MH116647 United States
CitationJournal: Nature / Year: 2020
Title: Electromechanical coupling in the hyperpolarization-activated K channel KAT1.
Authors: Michael David Clark / Gustavo F Contreras / Rong Shen / Eduardo Perozo /
Abstract: Voltage-gated potassium (K) channels coordinate electrical signalling and control cell volume by gating in response to membrane depolarization or hyperpolarization. However, although voltage-sensing ...Voltage-gated potassium (K) channels coordinate electrical signalling and control cell volume by gating in response to membrane depolarization or hyperpolarization. However, although voltage-sensing domains transduce transmembrane electric field changes by a common mechanism involving the outward or inward translocation of gating charges, the general determinants of channel gating polarity remain poorly understood. Here we suggest a molecular mechanism for electromechanical coupling and gating polarity in non-domain-swapped K channels on the basis of the cryo-electron microscopy structure of KAT1, the hyperpolarization-activated K channel from Arabidopsis thaliana. KAT1 displays a depolarized voltage sensor, which interacts with a closed pore domain directly via two interfaces and indirectly via an intercalated phospholipid. Functional evaluation of KAT1 structure-guided mutants at the sensor-pore interfaces suggests a mechanism in which direct interaction between the sensor and the C-linker hairpin in the adjacent pore subunit is the primary determinant of gating polarity. We suggest that an inward motion of the S4 sensor helix of approximately 5-7 Å can underlie a direct-coupling mechanism, driving a conformational reorientation of the C-linker and ultimately opening the activation gate formed by the S6 intracellular bundle. This direct-coupling mechanism contrasts with allosteric mechanisms proposed for hyperpolarization-activated cyclic nucleotide-gated channels, and may represent an unexpected link between depolarization- and hyperpolarization-activated channels.
History
DepositionNov 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 3, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 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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Structure visualization

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Assembly

Deposited unit
A: Potassium channel KAT1
B: Potassium channel KAT1
C: Potassium channel KAT1
D: Potassium channel KAT1
M: Potassium channel KAT1
N: Potassium channel KAT1
O: Potassium channel KAT1
P: Potassium channel KAT1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)484,40724
Polymers477,1178
Non-polymers7,29116
Water0
1


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

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Components

#1: Protein
Potassium channel KAT1


Mass: 59639.594 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: KAT1, At5g46240, MPL12.2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q39128
#2: Chemical
ChemComp-QNP / (2S)-1-(nonanoyloxy)-3-(phosphonooxy)propan-2-yl tetradecanoate


Mass: 522.652 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C26H51O8P
#3: Chemical
ChemComp-QNJ / (3beta,5beta,14beta,17alpha)-cholestan-3-ol / Coprostanol


Mass: 388.669 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C27H48O
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: Hyperpolarization-Activated Potassium Channel KAT1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPES1
2200 mMpotassium chlorideKCl1
32 mMcalcium chlorideCaCl21
40.05 %(w/v)digitonin1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1502
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION2particle selection
2Latitudeimage acquisition
4CTFFIND4CTF correction
7Cootmodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91689 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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