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- PDB-9oa8: Cryo-EM structure of KCa3.1/calmodulin channel in complex with NS309 -

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Basic information

Entry
Database: PDB / ID: 9oa8
TitleCryo-EM structure of KCa3.1/calmodulin channel in complex with NS309
Components
  • Calmodulin-1
  • Intermediate conductance calcium-activated potassium channel protein 4
KeywordsTRANSPORT PROTEIN / Ion channel / Intermediate conductance calcium-activated potassium channel / Calmodulin binding protein
Function / homology
Function and homology information


intermediate conductance calcium-activated potassium channel activity / saliva secretion / small conductance calcium-activated potassium channel activity / stabilization of membrane potential / Ca2+ activated K+ channels / macropinocytosis / calcium-activated potassium channel activity / transporter inhibitor activity / regulation of calcium ion import across plasma membrane / : ...intermediate conductance calcium-activated potassium channel activity / saliva secretion / small conductance calcium-activated potassium channel activity / stabilization of membrane potential / Ca2+ activated K+ channels / macropinocytosis / calcium-activated potassium channel activity / transporter inhibitor activity / regulation of calcium ion import across plasma membrane / : / positive regulation of potassium ion transmembrane transport / type 3 metabotropic glutamate receptor binding / positive regulation of T cell receptor signaling pathway / establishment of protein localization to membrane / cell volume homeostasis / phospholipid translocation / negative regulation of ryanodine-sensitive calcium-release channel activity / organelle localization by membrane tethering / response to corticosterone / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / negative regulation of calcium ion export across plasma membrane / regulation of synaptic vesicle exocytosis / regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle cell action potential / presynaptic endocytosis / calcineurin-mediated signaling / regulation of cell communication by electrical coupling involved in cardiac conduction / nitric-oxide synthase binding / adenylate cyclase binding / protein phosphatase activator activity / regulation of synaptic vesicle endocytosis / potassium channel activity / detection of calcium ion / immune system process / regulation of cardiac muscle contraction / postsynaptic cytosol / cell surface receptor signaling pathway via JAK-STAT / catalytic complex / phosphatidylinositol 3-kinase binding / calcium channel inhibitor activity / presynaptic cytosol / cellular response to interferon-beta / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / titin binding / regulation of calcium-mediated signaling / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / voltage-gated potassium channel complex / potassium ion transmembrane transport / calcium channel complex / regulation of heart rate / establishment of localization in cell / calyx of Held / nitric-oxide synthase regulator activity / adenylate cyclase activator activity / positive regulation of protein secretion / regulation of cytokinesis / response to amphetamine / protein serine/threonine kinase activator activity / spindle microtubule / sarcomere / positive regulation of receptor signaling pathway via JAK-STAT / calcium channel regulator activity / potassium ion transport / defense response / response to calcium ion / cellular response to type II interferon / G2/M transition of mitotic cell cycle / Schaffer collateral - CA1 synapse / ruffle membrane / spindle pole / calcium-dependent protein binding / calcium ion transport / myelin sheath / synaptic vesicle membrane / growth cone / sperm midpiece / protein phosphatase binding / vesicle / protein homotetramerization / transmembrane transporter binding / calmodulin binding / neuron projection / protein domain specific binding / neuronal cell body / calcium ion binding / centrosome / protein kinase binding / chromatin / protein homodimerization activity / protein-containing complex / mitochondrion / nucleoplasm / membrane / nucleus / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Calmodulin-binding domain / Potassium channel, calcium-activated, SK / SK, calmodulin-binding domain superfamily / Calmodulin binding domain / Calcium-activated SK potassium channel / Calmodulin binding domain / Potassium channel domain / Ion channel / : / EF-hand domain pair ...Calmodulin-binding domain / Potassium channel, calcium-activated, SK / SK, calmodulin-binding domain superfamily / Calmodulin binding domain / Calcium-activated SK potassium channel / Calmodulin binding domain / Potassium channel domain / Ion channel / : / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Chem-1KP / : / Intermediate conductance calcium-activated potassium channel protein 4 / Calmodulin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsNam, Y.W. / Zhang, M.
Funding support United States, 4items
OrganizationGrant numberCountry
American Heart Association23AIREA1039423 United States
American Heart Association24CDA1260237 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)4R33 NS101182-03 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R15 NS130420-01A1 United States
Citation
Journal: Nat Commun / Year: 2026
Title: Structural basis for the subtype-selectivity of K2.2 channel activators.
Authors: Young-Woo Nam / Alena Ramanishka / Yang Xu / Rose Marie Haynes Yasuda / Joshua A Nasburg / Dohyun Im / Meng Cui / K George Chandy / Heike Wulff / Miao Zhang /
Abstract: Small-conductance (K2.2) and intermediate-conductance (K3.1) Ca-activated K channels are gated by a Ca-calmodulin dependent mechanism. NS309 potentiates the activity of both K2.2 and K3.1, while ...Small-conductance (K2.2) and intermediate-conductance (K3.1) Ca-activated K channels are gated by a Ca-calmodulin dependent mechanism. NS309 potentiates the activity of both K2.2 and K3.1, while rimtuzalcap selectively activates K2.2. Rimtuzalcap has been used in clinical trials for the treatment of spinocerebellar ataxia and essential tremor. We report cryo-electron microscopy structures of NS309-bound K2.2 and K3.1, in addition to structures of rimtuzalcap-bound K2.2 and mutant K3.1_R355K. The different conformations of calmodulin and the cytoplasmic HC helices in the two channels underlie the subtype-selectivity of rimtuzalcap for K2.2. NS309 binds to pre-existing pockets in both channels, while the bulkier rimtuzalcap binds in an induced-fit pocket in K2.2 requiring conformational changes. In K2.2, calmodulin's N-lobes are sufficiently far apart to enable conformational changes to accommodate either NS309 or rimtuzalcap. In K3.1, calmodulin's N-lobes are closer to each other and constrained by K3.1's HC helices, which allows binding of NS309 but not rimtuzalcap. Replacement of arginine-355 in K3.1's HB helix with lysine (K3.1_R355K) allows the binding of rimtuzalcap and renders the mutant channel sensitive to rimtuzalcap. These structures provide a framework for structure-based drug design targeting K2.2 channels.
#1: Journal: Res Sq / Year: 2025
Title: Structural basis for the subtype-selectivity of K2.2 channel activators.
Authors: Miao Zhang / Young-Woo Nam / Alena Ramanishka / Yang Xu / Rose Marie Yasuda / Dohyun Im / Meng Cui / George Chandy / Heike Wulff /
Abstract: Small-conductance (K2.2) and intermediate-conductance (K3.1) Ca-activated K channels are gated by a Ca-calmodulin dependent mechanism. NS309 potentiates the activity of both K2.2 and K3.1, while ...Small-conductance (K2.2) and intermediate-conductance (K3.1) Ca-activated K channels are gated by a Ca-calmodulin dependent mechanism. NS309 potentiates the activity of both K2.2 and K3.1, while rimtuzalcap selectively activates K2.2. Rimtuzalcap has been used in clinical trials for the treatment of spinocerebellar ataxia and essential tremor. We report cryo-electron microscopy structures of K2.2 channels bound with NS309 and rimtuzalcap, in addition to K3.1 channels with NS309. The different conformations of calmodulin and the cytoplasmic HC helices in the two channels underlie the subtype-selectivity of rimtuzalcap for K2.2. Calmodulin's N-lobes in the K2.2 structure are far apart and undergo conformational changes to accommodate either NS309 or rimtuzalcap. Calmodulin's Nlobes in the K3.1 structure are closer to each other and are constrained by the HC helices of K3.1, which allows binding of NS309 but not of the bulkier rimtuzalcap. These structures provide a framework for structure-based drug design targeting K2.2 channels.
History
DepositionApr 19, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 18, 2025Provider: repository / Type: Initial release
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Revision 1.1Jun 25, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.title / _em_admin.last_update
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Revision 2.0Oct 1, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Refinement description
Category: atom_site / em_admin ...atom_site / em_admin / em_software / pdbx_struct_conn_angle / pdbx_validate_close_contact / pdbx_validate_torsion / refine_ls_restr / struct_conf / struct_conn
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Intermediate conductance calcium-activated potassium channel protein 4
B: Intermediate conductance calcium-activated potassium channel protein 4
C: Intermediate conductance calcium-activated potassium channel protein 4
D: Intermediate conductance calcium-activated potassium channel protein 4
E: Calmodulin-1
F: Calmodulin-1
G: Calmodulin-1
H: Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)237,84123
Polymers236,4798
Non-polymers1,36215
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Intermediate conductance calcium-activated potassium channel protein 4 / SKCa 4 / SKCa4 / hSK4 / Gardos channel / IKCa1 / hIK1 / KCa3.1 / Putative Gardos channel / hKCa4


Mass: 42598.633 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNN4, IK1, IKCA1, KCA4, SK4 / Production host: Homo sapiens (human) / References: UniProt: O15554
#2: Protein
Calmodulin-1


Mass: 16521.094 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Calm1, Calm, Cam, Cam1, CaMI / Production host: Homo sapiens (human) / References: UniProt: P0DP29
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-1KP / (3E)-6,7-dichloro-3-(hydroxyimino)-1,3-dihydro-2H-indol-2-one


Mass: 231.036 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H4Cl2N2O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Human KCa3.1/calmodulin channel in complex with NS309 / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.23166 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293s / Cell: HEK293 / Plasmid: pGEBacMam
Buffer solutionpH: 8
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2200 nm / Nominal defocus min: 1300 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
7Coot0.9.8.95 ELmodel fitting
9PHENIX1.21.1_5286model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119846 / Symmetry type: POINT
RefinementHighest resolution: 3.59 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00215520
ELECTRON MICROSCOPYf_angle_d0.40321029
ELECTRON MICROSCOPYf_dihedral_angle_d3.2862160
ELECTRON MICROSCOPYf_chiral_restr0.0352460
ELECTRON MICROSCOPYf_plane_restr0.0022641

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