|Entry||Database: PDB / ID: 6bgj|
|Title||Cryo-EM structure of the TMEM16A calcium-activated chloride channel in LMNG|
|Keywords||MEMBRANE PROTEIN / Chloride channel / TMEM16 family|
|Function/homology||Anoctamin-1 / Anoctamin, dimerisation domain / iodide transmembrane transporter activity / iodide transport / intracellular calcium activated chloride channel activity / Anoctamin / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Calcium-activated chloride channel / trachea development / calcium activated cation channel activity ...Anoctamin-1 / Anoctamin, dimerisation domain / iodide transmembrane transporter activity / iodide transport / intracellular calcium activated chloride channel activity / Anoctamin / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Calcium-activated chloride channel / trachea development / calcium activated cation channel activity / Stimuli-sensing channels / voltage-gated chloride channel activity / chloride channel activity / chloride channel complex / detection of temperature stimulus involved in sensory perception of pain / chloride transport / chloride transmembrane transport / cation transport / positive regulation of insulin secretion involved in cellular response to glucose stimulus / phospholipase C-activating G-protein coupled receptor signaling pathway / regulation of membrane potential / cellular response to heat / apical plasma membrane / external side of plasma membrane / protein heterodimerization activity / protein homodimerization activity / plasma membrane / cytoplasm / Anoctamin-1|
Function and homology information
|Specimen source||Mus musculus / mammal / House mouse /|
|Method||Electron microscopy (3.8 Å resolution / Particle / Single particle) / Transmission electron microscopy|
|Authors||Dang, S. / Feng, S. / Tien, J. / Peters, C.J. / Bulkley, D. / Lolicato, M. / Zhao, J. / Zuberbuhler, K. / Ye, W. / Qi, L. / Chen, T. / Craik, C.S. / Jan, Y.N. / Minor Jr., D.L. / Cheng, Y. / Jan, L.Y.|
|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 / 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.
SummaryFull reportAbout validation report
|Date||Deposition: Oct 28, 2017 / Release: Dec 27, 2017|
Downloads & links
Mass: 105603.984 Da / Num. of mol.: 2 / Details: Calcium ions / Source: (gene. exp.) Mus musculus / mammal / House mouse / / Gene: Ano1, Tmem16a / Production host: Homo sapiens / References: UniProt:Q8BHY3
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE|
|Component||Name: TMEM16A Calcium-activated chloride channel / Type: CELL / Entity ID: 1 / Source: RECOMBINANT|
|Source (natural)||Organism: Mus musculus|
|Source (recombinant)||Organism: Homo sapiens|
|Buffer solution||pH: 9|
|Specimen||Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins / Details: blot 6-8 seconds before plunging|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELD / Calibrated magnification: 29000 / Calibrated defocus min: 800 nm / Calibrated defocus max: 1900 nm / Cs: 2.7 mm / C2 aperture diameter: 100 mm / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 8 sec. / Electron dose: 80 e/Å2|
Details: 5326 images for TMEM16A alone in LMNG, 2556 images for TMEM16A with Fab bound in LMNG
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 7882
|Image scans||Movie frames/image: 40 / Used frames/image: 1-40|
|Software||Name: PHENIX / Version: 1.12_2829: / Classification: refinement|
|CTF correction||Type: NONE|
|Particle selection||Number of particles selected: 927414|
|Symmetry||Point symmetry: C2|
|3D reconstruction||Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 251851 / Algorithm: BACK PROJECTION / Number of class averages: 2 / Symmetry type: POINT|
|Atomic model building||Ref protocol: AB INITIO MODEL / Ref space: REAL|
|Refine LS restraints|
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