National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
DP2GM123496
United States
Citation
Journal: Nature / Year: 2020 Title: Structural basis for pH gating of the two-pore domain K channel TASK2. Authors: Baobin Li / Robert A Rietmeijer / Stephen G Brohawn / Abstract: TASK2 (also known as KCNK5) channels generate pH-gated leak-type K currents to control cellular electrical excitability. TASK2 is involved in the regulation of breathing by chemosensory neurons of ...TASK2 (also known as KCNK5) channels generate pH-gated leak-type K currents to control cellular electrical excitability. TASK2 is involved in the regulation of breathing by chemosensory neurons of the retrotrapezoid nucleus in the brainstem and pH homeostasis by kidney proximal tubule cells. These roles depend on channel activation by intracellular and extracellular alkalization, but the mechanistic basis for TASK2 gating by pH is unknown. Here we present cryo-electron microscopy structures of Mus musculus TASK2 in lipid nanodiscs in open and closed conformations. We identify two gates, distinct from previously observed K channel gates, controlled by stimuli on either side of the membrane. Intracellular gating involves lysine protonation on inner helices and the formation of a protein seal between the cytoplasm and the channel. Extracellular gating involves arginine protonation on the channel surface and correlated conformational changes that displace the K-selectivity filter to render it nonconductive. These results explain how internal and external protons control intracellular and selectivity filter gates to modulate TASK2 activity.
EMPIAR-10423 (Title: TASK2 in MSP1D1 lipid nanodisc at pH6.5 / Data size: 2.0 TB Data #1: multi-frame micrographs for ion channel TASK2 in closed state [micrographs - multiframe])
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Incubate 5 seconds, blot 3s seconds, blot force 1
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Electron microscopy imaging
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
Microscopy
Model: FEI TALOS ARCTICA
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holder
Cryogen: NITROGEN
Image recording
Average exposure time: 0.11 sec. / Electron dose: 0.98714 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2683
Image scans
Width: 11520 / Height: 8184
-
Processing
Software
Name
Version
Classification
NB
phenix.real_space_refine
1.16_3549
refinement
PHENIX
1.16_3549
refinement
EM software
ID
Name
Version
Category
1
RELION
3.1beta
particleselection
2
SerialEM
3-6
imageacquisition
4
CTFFIND
4.1
CTFcorrection
7
Coot
modelfitting
9
RELION
3.1beta
initialEulerassignment
10
RELION
3.1beta
finalEulerassignment
11
RELION
3.1beta
classification
12
RELION
3.1beta
3Dreconstruction
13
PHENIX
modelrefinement
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 3563337
Symmetry
Point symmetry: C1 (asymmetric)
3D reconstruction
Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 255808 / Num. of class averages: 1 / Symmetry type: POINT
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