National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM098672
United States
National Institutes of Health/Office of the Director
S100D0020054
United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
R01NS069229
United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)
R35NS097227
United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
R01HL080050
United States
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)
R01DC007664
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
CA196276
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM111126
United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)
K99DA041500
United States
Citation
Journal: Nature / Year: 2017 Title: Cryo-EM structures of the TMEM16A calcium-activated chloride channel. Authors: Shangyu Dang / Shengjie Feng / Jason Tien / Christian J Peters / David Bulkley / Marco Lolicato / Jianhua Zhao / Kathrin Zuberbühler / Wenlei Ye / Lijun Qi / Tingxu Chen / Charles S Craik / ...Authors: Shangyu Dang / Shengjie Feng / Jason Tien / Christian J Peters / David Bulkley / Marco Lolicato / Jianhua Zhao / Kathrin Zuberbühler / Wenlei Ye / Lijun Qi / Tingxu Chen / Charles S Craik / Yuh Nung Jan / Daniel L Minor / Yifan Cheng / Lily Yeh Jan / Abstract: Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the ...Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.
History
Deposition
Oct 28, 2017
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Header (metadata) release
Dec 27, 2017
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Map release
Dec 27, 2017
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Update
Mar 13, 2024
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Current status
Mar 13, 2024
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
EMPIAR-10123 (Title: Cryo-EM structure of the TMEM16A in Nanodisc / Data size: 226.7 Data #1: Particle stacks extracted from motion corrected micrographs of TMEM16A in nanodisc [picked particles - single frame - processed])
Name: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA
Molecular weight
Theoretical: 40.078 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
0.5 mg/mL
Buffer
pH: 9 Component:
Concentration
Formula
Name
10.0 mM
TrisNO3
Tris Nitrate
150.0 mM
KNO3
Potassium Nitrate
1.0 mM
CaCl2
Calcium Chloride
Grid
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.027 kPa
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: blot 6-8 seconds before plunging.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 3149 / Average exposure time: 8.0 sec. / Average electron dose: 80.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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