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- PDB-9czk: Ca2+ free hSlo1 + beta2N-beta4 channel in nanodisc. -

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Basic information

Entry
Database: PDB / ID: 9czk
TitleCa2+ free hSlo1 + beta2N-beta4 channel in nanodisc.
Components
  • Isoform 5 of Calcium-activated potassium channel subunit alpha-1
  • Large-conductance Ca2+-activated K+ channel beta2 subunit,Calcium-activated potassium channel subunit beta-4
KeywordsMEMBRANE PROTEIN / Potassium ion channel / calcium and voltage gated ion channel / big potassium channel / human BK / hSlo1 / apo hSlo1 / closed hSlo1 / ball and chain inactivation / hSlo1 inactivating subunit complex / nanodisc
Function / homology
Function and homology information


Acetylcholine inhibits contraction of outer hair cells / micturition / large conductance calcium-activated potassium channel activity / Ca2+ activated K+ channels / calcium-activated potassium channel activity / negative regulation of cell volume / regulation of neurotransmitter secretion / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / smooth muscle contraction involved in micturition / response to carbon monoxide ...Acetylcholine inhibits contraction of outer hair cells / micturition / large conductance calcium-activated potassium channel activity / Ca2+ activated K+ channels / calcium-activated potassium channel activity / negative regulation of cell volume / regulation of neurotransmitter secretion / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / smooth muscle contraction involved in micturition / response to carbon monoxide / response to osmotic stress / Sensory processing of sound by inner hair cells of the cochlea / cGMP effects / intracellular potassium ion homeostasis / action potential / voltage-gated potassium channel activity / detection of calcium ion / potassium channel regulator activity / regulation of vasoconstriction / neuronal action potential / voltage-gated potassium channel complex / potassium ion transmembrane transport / regulation of membrane potential / response to calcium ion / caveola / potassium ion transport / vasodilation / actin binding / monoatomic ion transmembrane transport / chemical synaptic transmission / postsynaptic membrane / response to hypoxia / positive regulation of apoptotic process / apical plasma membrane / synapse / metal ion binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
KCNMB2, ball/chain domain / KCNMB2, ball/chain domain superfamily / KCNMB2, ball and chain domain / Potassium channel, calcium-activated, BK, beta subunit / Calcium-activated potassium channel, beta subunit / : / Ca2+-activated K+ channel Slowpoke, TrkA_C like domain / Calcium-activated potassium channel BK, alpha subunit / : / Calcium-activated BK potassium channel alpha subunit ...KCNMB2, ball/chain domain / KCNMB2, ball/chain domain superfamily / KCNMB2, ball and chain domain / Potassium channel, calcium-activated, BK, beta subunit / Calcium-activated potassium channel, beta subunit / : / Ca2+-activated K+ channel Slowpoke, TrkA_C like domain / Calcium-activated potassium channel BK, alpha subunit / : / Calcium-activated BK potassium channel alpha subunit / Calcium-activated potassium channel slowpoke-like RCK domain / Regulator of K+ conductance, N-terminal / RCK N-terminal domain profile. / Ion transport domain / Ion transport protein / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
CHOLESTEROL / : / Chem-POV / Calcium-activated potassium channel subunit beta-2 / Calcium-activated potassium channel subunit alpha-1 / Calcium-activated potassium channel subunit beta-4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsAgarwal, S. / Nimigean, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR) United States
CitationJournal: Nat Commun / Year: 2025
Title: Ball-and-chain inactivation of a human large conductance calcium-activated potassium channel.
Authors: Shubhangi Agarwal / Elizabeth D Kim / Sangyun Lee / Alexander Simon / Alessio Accardi / Crina M Nimigean /
Abstract: BK channels are large-conductance calcium (Ca)-activated potassium channels crucial for neuronal excitability, muscle contraction, and neurotransmitter release. The pore-forming (α) subunits co- ...BK channels are large-conductance calcium (Ca)-activated potassium channels crucial for neuronal excitability, muscle contraction, and neurotransmitter release. The pore-forming (α) subunits co-assemble with auxiliary (β and γ) subunits that modulate their function. Previous studies demonstrated that the N-termini of β2-subunits can inactivate BK channels, but with no structural correlate. Here, we investigate BK β2-subunit inactivation using cryo-electron microscopy, electrophysiology and molecular dynamics simulations. We find that the β2 N-terminus occludes the pore only in the Ca-bound open state, via a ball-and-chain mechanism. The first three hydrophobic residues of β2 are crucial for occlusion, while the remainder of the N-terminus remains flexible. Neither the closed channel conformation obtained in the absence of Ca nor an intermediate conformation found in the presence of Ca show density for the N-terminus of the β2 subunit in their pore, likely due to narrower side access portals preventing their entry into the channel pore.
History
DepositionAug 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Isoform 5 of Calcium-activated potassium channel subunit alpha-1
B: Isoform 5 of Calcium-activated potassium channel subunit alpha-1
C: Isoform 5 of Calcium-activated potassium channel subunit alpha-1
D: Isoform 5 of Calcium-activated potassium channel subunit alpha-1
E: Large-conductance Ca2+-activated K+ channel beta2 subunit,Calcium-activated potassium channel subunit beta-4
F: Large-conductance Ca2+-activated K+ channel beta2 subunit,Calcium-activated potassium channel subunit beta-4
G: Large-conductance Ca2+-activated K+ channel beta2 subunit,Calcium-activated potassium channel subunit beta-4
H: Large-conductance Ca2+-activated K+ channel beta2 subunit,Calcium-activated potassium channel subunit beta-4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)590,27118
Polymers585,6068
Non-polymers4,66510
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Isoform 5 of Calcium-activated potassium channel subunit alpha-1 / BK channel / BKCA alpha / Calcium-activated potassium channel / subfamily M subunit alpha-1 / K(VCA) ...BK channel / BKCA alpha / Calcium-activated potassium channel / subfamily M subunit alpha-1 / K(VCA)alpha / KCa1.1 / Maxi K channel / MaxiK / Slo-alpha / Slo1 / Slowpoke homolog / Slo homolog / hSlo


Mass: 118969.945 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNMA1, KCNMA, SLO / Production host: Homo sapiens (human) / References: UniProt: Q12791
#2: Protein
Large-conductance Ca2+-activated K+ channel beta2 subunit,Calcium-activated potassium channel subunit beta-4 / BK channel subunit beta-4 / BKbeta4 / Hbeta4 / Calcium-activated potassium channel / subfamily M ...BK channel subunit beta-4 / BKbeta4 / Hbeta4 / Calcium-activated potassium channel / subfamily M subunit beta-4 / Charybdotoxin receptor subunit beta-4 / K(VCA)beta-4 / Maxi K channel subunit beta-4 / Slo-beta-4


Mass: 27431.523 Da / Num. of mol.: 4
Fragment: N-terminal 45 residues of kcnmb2 ligated to kcnmb4 (devoid of N terminal first 15 residues),N-terminal 45 residues of kcnmb2 ligated to kcnmb4 (devoid of N terminal first 15 residues)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNMB2, KCNMB4 / Production host: Homo sapiens (human) / References: UniProt: B5BNX0, UniProt: Q86W47
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#5: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSource
1Ca2+ free BK channel complex of hSlo1 + beta2N-beta4 subunitCOMPLEX#1-#20RECOMBINANT
2Inactivating beta2N-beta4 subunit chimeraCOMPLEX#21RECOMBINANT
3hSlo1 channelCOMPLEX#11RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Buffer solutionpH: 8
Details: 20 mM Tris-HCl pH 8.0, 450 mM KCl, 5 mM EDTA, 15 mM MgCl2, 0.02% GDN and 0.05 mg/ml POPE:POPC:POPA 5:5:1 (w:w:w).
SpecimenConc.: 9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 47.08 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2Leginon3.7image acquisition
4RELION4.0.0CTF correction
7Coot0.8.9model fitting
9PHENIX1.21.1model refinement
10RELION4.0.0initial Euler assignment
11RELION4.0.0final Euler assignment
12RELION4.0.0classification
13RELION4.0.03D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58904 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6v35

6v35


Accession code: 6v35 / Source name: PDB / Type: experimental model

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