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- PDB-6xev: CryoEM structure of GIRK2-PIP2/CHS - G protein-gated inwardly rec... -

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Basic information

Entry
Database: PDB / ID: 6xev
TitleCryoEM structure of GIRK2-PIP2/CHS - G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2
ComponentsG protein-activated inward rectifier potassium channel 2
KeywordsTRANSPORT PROTEIN / CryoEM / GIRK / inwardly rectifying potassium channel / cholesterol / lipids / PIP2
Function / homology
Function and homology information


G-protein activated inward rectifier potassium channel activity / monoatomic ion channel complex
Similarity search - Function
Potassium channel, inwardly rectifying, Kir3.2 / 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 / Immunoglobulin E-set
Similarity search - Domain/homology
: / Chem-PIO / CHOLESTEROL HEMISUCCINATE / G protein-activated inward rectifier potassium channel 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMathiharan, Y.K. / Glaaser, I.W. / Skiniotis, G. / Slesinger, P.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (NIH/NIAAA)AA018734 United States
CitationJournal: Cell Rep / Year: 2021
Title: Structural insights into GIRK2 channel modulation by cholesterol and PIP.
Authors: Yamuna Kalyani Mathiharan / Ian W Glaaser / Yulin Zhao / Michael J Robertson / Georgios Skiniotis / Paul A Slesinger /
Abstract: G-protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability. In addition to G proteins, GIRK channels are potentiated by membrane cholesterol, ...G-protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability. In addition to G proteins, GIRK channels are potentiated by membrane cholesterol, which is elevated in the brains of people with neurodegenerative diseases such as Alzheimer's dementia and Parkinson's disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. In this study, we present cryo- electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and phosphatidylinositol 4,5-bisphosphate (PIP). The structures reveal that CHS binds near PIP in lipid-facing hydrophobic pockets of the transmembrane domain. Our structural analysis suggests that CHS stabilizes PIP interaction with the channel and promotes engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of one of the CHS binding pockets eliminates cholesterol-dependent potentiation of GIRK2. Elucidating the structural mechanisms underlying cholesterol modulation of GIRK2 channels could facilitate the development of therapeutics for treating neurological diseases. VIDEO ABSTRACT.
History
DepositionJun 14, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: G protein-activated inward rectifier potassium channel 2
E: G protein-activated inward rectifier potassium channel 2
I: G protein-activated inward rectifier potassium channel 2
M: G protein-activated inward rectifier potassium channel 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,78129
Polymers156,2484
Non-polymers5,53325
Water0
1


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

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Components

#1: Protein
G protein-activated inward rectifier potassium channel 2


Mass: 39061.957 Da / Num. of mol.: 4 / Fragment: UNP residues 34-362
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Kcnj6 / Production host: Komagataella pastoris (fungus) / References: UniProt: A0A338P6L0
#2: Chemical
ChemComp-PIO / [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C25H49O19P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: K
#5: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na
Has ligand of interestY

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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: G protein-gated inwardly rectifying potassium channel (GIRK2)
Type: COMPLEX
Details: CryoEM structure of G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2
Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
2150 mMPotassium ChlorideKCl1
320 mMDTTC4H10O2S21
43 mMTCEPC9H15O6P1
51 mMEDTAEthylenediaminetetraacetic acidC10H16N2O81
60.025 %DDMC24H46O111
70.0025 %CHSC31H50O41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: CryoEM structure G protein-gated inwardly rectifying potassium channel GIRK2 with modulators cholesteryl hemisuccinate and PIP2
Specimen supportGrid material: GOLD / 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: 293 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 FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 83 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
14RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 297174
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44990 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 3SYA

3sya

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