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- PDB-8ti1: Cryo-EM structure of a SUR1/Kir6.2-Q52R ATP-sensitive potassium c... -

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Basic information

Entry
Database: PDB / ID: 8ti1
TitleCryo-EM structure of a SUR1/Kir6.2-Q52R ATP-sensitive potassium channel in the presence of PIP2 in the open conformation
Components
  • ATP-sensitive inward rectifier potassium channel 11
  • SUR1
KeywordsTRANSPORT PROTEIN / ATP-sensitive potassium channel / KATP channel / SUR1 / Kir6.2-Q52R / potassium transport / metabolic sensor / diabetes / phospholipid binding / PIP2
Function / homology
Function and homology information


Regulation of insulin secretion / ATP sensitive Potassium channels / ABC-family proteins mediated transport / response to resveratrol / ATP-activated inward rectifier potassium channel activity / inward rectifying potassium channel / sulfonylurea receptor activity / cell body fiber / ventricular cardiac muscle tissue development / CAMKK-AMPK signaling cascade ...Regulation of insulin secretion / ATP sensitive Potassium channels / ABC-family proteins mediated transport / response to resveratrol / ATP-activated inward rectifier potassium channel activity / inward rectifying potassium channel / sulfonylurea receptor activity / cell body fiber / ventricular cardiac muscle tissue development / CAMKK-AMPK signaling cascade / voltage-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / ATPase-coupled monoatomic cation transmembrane transporter activity / inward rectifier potassium channel activity / regulation of monoatomic ion transmembrane transport / nervous system process / inorganic cation transmembrane transport / response to stress / ankyrin binding / Ion homeostasis / response to ATP / potassium ion import across plasma membrane / response to testosterone / action potential / voltage-gated potassium channel activity / intercalated disc / axolemma / ABC-type transporter activity / negative regulation of insulin secretion / cellular response to nutrient levels / heat shock protein binding / T-tubule / potassium ion transmembrane transport / regulation of insulin secretion / acrosomal vesicle / regulation of membrane potential / response to ischemia / determination of adult lifespan / positive regulation of protein localization to plasma membrane / cellular response to glucose stimulus / potassium ion transport / sarcolemma / cellular response to nicotine / glucose metabolic process / cellular response to tumor necrosis factor / response to estradiol / nuclear envelope / presynaptic membrane / transmembrane transporter binding / response to hypoxia / endosome / response to xenobiotic stimulus / neuronal cell body / glutamatergic synapse / apoptotic process / ATP hydrolysis activity / protein-containing complex / ATP binding / plasma membrane / cytoplasm
Similarity search - Function
Potassium channel, inwardly rectifying, Kir6.2 / ATP-binding cassette subfamily C member 8 / Sulphonylurea receptor / Potassium channel, inwardly rectifying, transmembrane domain / Inward rectifier potassium channel transmembrane domain / Potassium channel, inwardly rectifying, Kir, cytoplasmic / Potassium channel, inwardly rectifying, Kir / Inward rectifier potassium channel, C-terminal / Inward rectifier potassium channel C-terminal domain / : ...Potassium channel, inwardly rectifying, Kir6.2 / ATP-binding cassette subfamily C member 8 / Sulphonylurea receptor / Potassium channel, inwardly rectifying, transmembrane domain / Inward rectifier potassium channel transmembrane domain / Potassium channel, inwardly rectifying, Kir, cytoplasmic / Potassium channel, inwardly rectifying, Kir / Inward rectifier potassium channel, C-terminal / Inward rectifier potassium channel C-terminal domain / : / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / Immunoglobulin E-set / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / Chem-P5S / DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / Chem-PT5 / SUR1 / ATP-sensitive inward rectifier potassium channel 11
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Mesocricetus auratus (golden hamster)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsDriggers, C.M. / Shyng, S.-L.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK066485 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129547 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structure of an open K channel reveals tandem PIP binding sites mediating the Kir6.2 and SUR1 regulatory interface.
Authors: Camden M Driggers / Yi-Ying Kuo / Phillip Zhu / Assmaa ElSheikh / Show-Ling Shyng /
Abstract: ATP-sensitive potassium (K) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. K ...ATP-sensitive potassium (K) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. K channel opening is stimulated by PIP and inhibited by ATP. Mutations that increase channel opening by PIP reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP in K channel function, previously solved open-channel structures have lacked bound PIP, and mechanisms by which PIP regulates K channels remain unresolved. Here, we report the cryoEM structure of a K channel harboring the neonatal diabetes mutation Kir6.2-Q52R, in the open conformation, bound to amphipathic molecules consistent with natural C18:0/C20:4 long-chain PI(4,5)P at two adjacent binding sites between SUR1 and Kir6.2. The canonical PIP binding site is conserved among PIP-gated Kir channels. The non-canonical PIP binding site forms at the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP binding and gating, explain the antagonistic regulation of K channels by PIP and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.
History
DepositionJul 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ATP-sensitive inward rectifier potassium channel 11
E: SUR1
B: ATP-sensitive inward rectifier potassium channel 11
C: ATP-sensitive inward rectifier potassium channel 11
D: ATP-sensitive inward rectifier potassium channel 11
H: SUR1
G: SUR1
F: SUR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)902,47236
Polymers883,9508
Non-polymers18,52228
Water25214
1


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

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Components

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Protein , 2 types, 8 molecules ABCDEHGF

#1: Protein
ATP-sensitive inward rectifier potassium channel 11 / BIR / Inward rectifier K(+) channel Kir6.2 / Potassium channel / inwardly rectifying subfamily J member 11


Mass: 43690.828 Da / Num. of mol.: 4 / Mutation: Q52R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Cell: Beta cell / Gene: Kcnj11 / Organ: Pancreas / Cell line (production host): COS-M6 (RRID:CVCL_8561) / Production host: Chlorocebus aethiops (grivet) / References: UniProt: P70673
#2: Protein
SUR1


Mass: 177296.578 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mesocricetus auratus (golden hamster) / Cell line (production host): COS-M6 (RRID:CVCL_8561) / Production host: Chlorocebus aethiops (grivet) / References: UniProt: A0A1S4NYG1

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Sugars , 1 types, 4 molecules

#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 5 types, 38 molecules

#3: Chemical
ChemComp-PT5 / [(2R)-1-octadecanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phospho ryl]oxy-propan-2-yl] (8Z)-icosa-5,8,11,14-tetraenoate / Phosphatidylinositol 4,5-bisphosphate / PtdIns(4,5)P2


Mass: 1047.088 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C47H85O19P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#4: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: K
#5: Chemical
ChemComp-PEF / DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / 3-[AMINOETHYLPHOSPHORYL]-[1,2-DI-PALMITOYL]-SN-GLYCEROL


Mass: 691.959 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C37H74NO8P / Comment: phospholipid*YM
#6: Chemical
ChemComp-P5S / O-[(R)-{[(2R)-2,3-bis(octadecanoyloxy)propyl]oxy}(hydroxy)phosphoryl]-L-serine / phosphatidyl serine


Mass: 792.075 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C42H82NO10P
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Formula: H2O

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Details

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

ComponentName: Kir6.2-Q52R/SUR1 open channel / Type: COMPLEX
Details: Kir6.2-Q52R/SUR1 open KATP channel in the open conformation in complex with PIP2 and other phospholipids
Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.880 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Chlorocebus aethiops (grivet) / Cell: COS-M6 cells / Plasmid: recombinant adenovirus
Buffer solutionpH: 7.4 / Details: MSB with digitonin and PIP2
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
2100 mMpotassium chlorideKCl1
350 mMHEPES pH 7.51
41 mMbrain Phosphatidylinositol 4,5-bisphosphate1
50.05 %digitonin1
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 3 microliters of purified Kir6.2-Q52R/FLAG-SUR1 was loaded onto Quantifoil R 1.2/1.3 Au 300 grids prepared with a fresh Graphene Oxide surface
Specimen supportDetails: The grid was prepared with a Graphene Oxide coating before use.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Titan Krios #3 at the Pacific Northwest National Lab
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.2 sec. / Electron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 3 / Num. of real images: 5241

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
4cryoSPARC4.2.1CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC4.2.1initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
12cryoSPARC4.2.1classification
13cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14115
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14115 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 6BAA

6baa


Accession code: 6BAA / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00251164
ELECTRON MICROSCOPYf_angle_d0.43269844
ELECTRON MICROSCOPYf_dihedral_angle_d11.28317032
ELECTRON MICROSCOPYf_chiral_restr0.0398516
ELECTRON MICROSCOPYf_plane_restr0.0038772

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